Probioti

See
discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/47931788
Probiotic activity of Lactobacillus delbrueckii

subsp. bulgaricus in the oral cavity
Article
Source: OAI
CITATIONS
READS
106
1 author:
Iva Stamatova
Medical University of Plovdiv
13 PUBLICATIONS 281 CITATIONS
SEE PROFILE
All content following this page was uploaded by Iva Stamatova on 02 December 2016.
The user has requested enhancement of the downloaded file. All in-text references underlined in blue
are linked to publications on ResearchGate, letting you access and read them immediately.
Institute of Dentistry, Biomedicum Research Laboratory,

University of Helsinki, and the Department of Oral and Maxillofacial Diseases,
Helsinki University Central Hospital, Finland
Probiotic activity of Lactobacillus delbrueckii

subsp. bulgaricus in the oral cavity
An in vitro study
Iva Vaseva Stamatova
ACADEMIC DISSERTATION
To be presented, with the permission of the Medical Faculty of the University of
Helsinki, for public examination in Lecture hall 3 of Biomedicum, Haartmaninkatu 8,
on 24 March 2010, at 12 oclock noon.
Helsinki 2010
ISBN 978-952-10-6152-3 (pbk.)

ISBN 978-952-10-6153-0 (PDF)
Helsinki University Print
Helsinki 2010
Supervised by:
Professor Jukka H. Meurman, MD, PhD, DOdont

Institute of Dentistry
University of Helsinki
and Department of Oral and
Maxillofacial Diseases
Helsinki University Central Hospital,
Helsinki, Finland
Co-supervized:
Professor Maria Baltadjieva, PhD

LB Lact, Plovdiv, Bulgaria
Professor Stoyan Vladimirov, DDS, PhD
Faculty of Dental Medicine, Medical University,
Plovdiv, Bulgaria
Reviewed by:
Professor Jorma Tenovuo, DDS, PhD

Department of Cariology,
Institute of Dentistry, University of Turku and
Department of Oral Diseases,
Turku University Central Hospital,
Turku, Finland
Professor Riita Korpela, PhD

Institute of Biomedicine, Pharmacology
University of Helsinki,
Helsinki, Finland
Opponent:
Professor Seppo Salminen, PhD

Functional Foods Forum
University of Turku
Turku, Finland
Abstract
The long established tradition of yogurt consumption has been related to longevity of
some populations living on the Balkans. Yogurt starter L. delbrueckii subsp. bulgaricus
and Str. thermophilus have recently been recognized as probiotics with verified beneficial
health effects. The oral cavity emerges as a relevant target for probiotic applications and
probiotics have demonstrated promising results in controlling dental diseases and yeast
infections. However, L. delbrueckii subsp. bulgaricus despite its broad availability in
fermented dairy products has not been evaluated for possible probiotic activity in the
mouth.
These series of studies were conducted to investigate in vitro properties of L.
delbrueckii subsp. bulgaricus to outline its potential as an oral probiotic. Prerequisite
probiotic properties in the oral cavity are resistance to oral defense mechanisms,
adherence to saliva-coated surfaces, and inhibition of oral pathogens. L. delbrueckii subsp.
bulgaricus strains showed a strain-dependent inhibition of oral streptococci and
Aggregatibacter actinomycetemcomitans, whereas none of the dairy starter strains could
affect growth of Porphyromonas gingivalis and Fusobacterium nucleatum.
Adhesion to surfaces is a factor contributing to prolonged establishment of the species
at the target site. Fifteen radiolabeled dairy L. delbrueckii subsp. bulgaricus strains and L.
rhamnosus GG were tested for their ability to adhere to saliva-coated hydroxyapatite
beads and polystyrene microtiter plates. The effects of lysozyme on the adhesion of
lactobacilli and of the pretreatment with lactobacilli on the adhesion of Streptococcus
sanguinis were also assessed. The adhesion of the L. delbrueckii subsp. bulgaricus strains
remained lower in comparison to L. rhamnosus GG. One L. delbrueckii subsp. bulgaricus
strain showed binding frequency comparable to S. sanguinis. Lysozyme pretreatment of
the samples significantly increased Lactobacillus adhesion to saliva-coated surfaces.
Insubstantially low gelatinolytic activity was observed in the supernatant and cell
fractions of all strains supernatant specimens being slightly more proteolytic, and no
conversion of proMMP-9 to its active form was induced by L. delbrueckii subsp.
bulgaricus. Safety assessment ruled out deleterious effects of L. delbrueckii subsp.
bulgaricus on extracellular matrix structures.
Cytokine response of oral epithelial cells after L. delbrueckii subsp. bulgaricus
challenge was assessed by measuring IL-8 and TNF- levels in cell culture supernatants.
The effect of Porphyromonas gingivalis on cytokine secretion after lactobacilli
pretreatment was also assessed. A strain- and time-dependent induction of IL-8 was
observed with live bacteria inducing the highest levels of cytokine secretion. Generally,
levels of TNF- were low and only one of ten L. delbrueckii subsp. bulgaricus strains
stimulated TNF- secretion closely to that of the positive control. The addition of P.
gingivalis produced almost an immediate reduction of cytokine levels within the first
hours of incubation irrespective of lactobacilli strains co-cultured with epithelial cells.
According to this series of studies there are strains among the L. delbrueckii subsp.
bulgaricus species that may have beneficial probiotic properties in the human oral cavity
and their potential in prevention and management of common oral infectious diseases to
be further studied.
Contents
Abstract
List of original publications
Abbreviations
10
1 Introduction
11
2 Review of the literature
12
2.1 Oral cavity in health and disease
12
Key elements of oral homeostasis
12
Oral mucosa
12
Saliva
13
Oral microbiota
13
2.2 Common oral diseases

2.2.1 Dental caries
14
14
2.2.1.1 Etiology and pathogenesis
14
2.2.1.2 Treatment and prevention
16
2.2.2 Periodontal disease
17
17
18
2.3 Probiotics from the oral health perspective
19
2.3.1 Probiotics
19
2.3.1.1 Emergence and definition of the term
19
2.3.1.2 Beneficial effects of probiotics
20
2.3.1.3 Selection criteria for probiotic candidates
23
2.3.1.3.1 Attachment, adhesion, and oral colonization
24
2.3.1.3.2 Resistance to oral defence mechanisms
26
2.3.2 Clinical relevance of probiotics in the oral cavity
26
2.3.2.1 Probiotics and dental caries
28
2.3.2.2 Probiotics and periodontal disease
28
2.3.2.3 Probiotics and other oral disorders
29
2.4 L. delbrueckii subsp. bulgaricus as a probiotic
30
2.5 Issues of safety in the oral health perspective
34
3 Hypotheses and aims of the study
36
4 Materials and methods
37
4.1 Bacterial strains and culture conditions
37
4.1.1 Lactic acid bacteria
37
4.1.2 Oral bacteria
39
4.1.3 Cell cultures
41
4.2 Methods
41
4.3 Study design
42
4.3.1 Antimicrobial activity against various oral pathogens (I)
42
4.3.2 Adhesion to saliva-coated surfaces in vitro (II)
42
4.3.2.1 Adhesion to sHA beads
43
4.3.2.2 Adhesion to saliva-coated microtiter plates
43
4.3.2.3 Adhesion to solvents
43
4.3.2.4 Effect of lactobacilli pretreatment on streptococcal adhesion in

vitro
43
4.3.3 Proteolytic activity on human gelatinases (III)
44
4.3.4 Epithelial cell response to L. delbrueckii subsp. bulgaricus
44
4.3.4.1 Induction of IL-8 and TNF- secretion
44
4.3.4.2 Epithelial cell response to P. gingivalis after lactobacilli

pretreatment
44
4.4 Statistical analyses
45
5 Results and discussion
46
5.1 Inhibition of oral pathogens (Study I)
46
5.2 L. bulgaricus adhesion to saliva-coated surfaces (Study II)
49
5.3 Proteolytic activity on human progelatinase B (proMMP-9) (Study III)
51
5.3.1 Gelatinolytic activity
51
5.3.2 Activation of proMMP-9
52
5.3.2 Proteolytic activity of L. bulgaricus strains in the presence of synthetic

MMP inhibitors
54
5.4 Epithelial cell lactobacilli interactions (Study IV)
54
6 Key findings and conclusions
57
Acknowledgements
58
References
60
List of original publications

This thesis is based on the following original articles referred to in the text by their
Roman numerals I - IV:
Stamatova I, Kari K, Meurman JH. In vitro evaluation of antimicrobial

activity of putative probiotic lactobacilli against oral pathogens. Int J
Probiotics Prebiotics 2007;2:225-232.
II
Stamatova I, Kari K, Vladimirov S, Meurman JH. In vitro evaluation of

yoghurt starter lactobacilli and Lactobacillus rhamnosus GG adhesion to
saliva-coated surfaces. Oral Microbiol Immunol 2009;24:218-223.
III
Stamatova I, Meurman JH, Kari K, Tervahartiala T, Sorsa T,

Baltadjieva M. Safety issues of Lactobacillus bulgaricus with respect to
human gelatinases in vitro. FEMS Immunol Med Microbiol. 2007;51:194200.
IV
Stamatova I, Kari K, Meurman JH. Probiotic challenge of oral epithelial

cells in vitro. Int J Probiotics Prebiotics (accepted).
The publications are referred to in the text by their Roman numerals.

The original articles are reprinted with the kind permission of the copyright holders.
Abbreviations
ATCC
American type culture collection
CFU
Ccolony forming unit
DNA
Deoxyribonucleic acid
ECM
Extra-cellular matix
EIR
Effective inhibition ratio
ELISA
Enzyme linked immunosorbent assay
GCF
Gingival crevicular fluid
GIT
Gastrointestinal tract
GRAS
Generally regarded as safe
HA
Hydroxyapatite
HDP
Host-defense peptide
IL-8
Interleukin - 8
LAB
Lactic acid bacteria
LDL
Low density lipoprotein
MRS
De Man, Rogosa and Sharpe medium
OD
Optical density
PBS
Phosphate buffered saline
RNA
Ribonucleic acid
SDS
Sodium dodecyl sulphate
SDS-PAGE
Sodium dodecyl sulphate

polyacrylamide gel electrophoresis
TLR
Toll-like receptor
TNF-
Tumor necrosis factor -
VSC
Volatile sulphur compounds
Microliter
10
1 Introduction
In recent decades, probiotic applications have emerged as a fascinating strategy to
alleviate symptoms of various diseases, predominantly in the gastrointestinal tract.
Probiotics are defined as live microorganisms conferring health benefit on the host when
administered in sufficient amount (www.who.int/foodsafety/fs_management/en/probiotic
_guidelines.pdf). In humans, the most frequently used probiotics are bacteria from the
genera Lactobacillus or Bifidobacterium. The list of probiotic species tend to increase and
new strains to be upended. The efficacy of L. delbrueckii subsp. bulgaricus as a probiotic
has been questionable due to inconclusive evidence of its establishment and survival in the
gastrointestinal tract. However, with the accumulation of new data and because of its
ubiquitous availability in fermented dairy products, the yogurt starter L. delbrueckii subsp.
bulgaricus was recognized as probiotic when a health benefit was validated in clinical
trials (Guarner et al., 2005).
The oral cavity as a gateway to the underlying gastrointestinal tract is the first site of
contact between probiotics and the host. Despite structural similarities with other parts of
the digestive system oral cavity is unique for its highly specialized functions and
characteristic site-specific pathology. The most common oral diseases with great social
repercussions remain to be dental caries and periodontal disease. The infectious etiology
of both these pathological conditions is well established and various strategies for control
of pathogenic oral biofilms are in use.
The idea that probiotic administration may improve some disease conditions in the
mouth has recently been introduced and the number of studies published gradually
increase (aglar et al., 2005; Meurman, 2005). Among the probiotics used in the oral
cavity are species such as L. rhamnosus GG, L. reuteri, L. casei, B. lactis that have shown
to different extent capacity to reduce mutans streptococci counts or lessen gingival
inflammation. However, the precise mechanisms explaining the observed effects yet
remain unclear.
To comply with the term probiotic several basic requirements should be considered.
Among these are: 1) safety of the microorganism; 2) conferring health benefits; 3)
adhesion and colonization capacity; 4) inhibition of pathogens; 5) survival and resistance
to human defense mechanisms. Additionally, in the scope of the oral cavity, probiotic
carbohydrate and protein utilization patterns should not expose oral structures to risk of
disease such as caries.
Whenever a new probiotic candidate is evaluated a number of basic in vitro screening
tests are used. Although in vitro studies have limited ability to completely reproduce
authentic environmental conditions, they are essential steps in discovery of species that
may further be used in clinical settings.
The millennium long tradition of yogurt consumption and the GRAS (generally
regarded as safe) status of lactobacilli encouraged us to conduct this series of studies;
evaluating the L. delbrueckii subsp. bulgaricus strains in its inhibitory capacity against
common oral pathogens, adherence to saliva-coated surfaces, interaction with oral
epithelium and above all to test its harmlessness to oral structures.
11
2 Review of the literature
2.1 Oral cavity in health and disease

The oral cavity as an integral part of the digestive system has various specific functions.
Although it is the entry and part of the gastrointestinal tract (GIT), it possesses some
distinctive features that makes it different from the rest of the digestive system. Mouth is a
unique complex system of tissues and organs that together are involved in nutritional,
respiratory, and communicative functions.
Oral health is important for overall health which requires harmonious functioning of
several key elements in the mouth. The impact of common oral diseases extends beyond
the oral cavity (Thorstensson and Johansson, 2009). Oral infection has been found to be
associated with death risk in studies among middle-aged individuals (DeStefano et al.,
1993; Garcia et al., 1998; Soikkonen et al., 2000; Jansson et al., 2002). The relative
importance of oral health as a predictor of survival has also been analyzed and the
common oral diseases have shown significant influence on survival (sterberg et al.,
1990; Cabrera et al., 2005; Semba et al., 2006; Morita et al., 2006). Therefore, keeping
oral health unperturbed and well balanced predisposes to long term and stable well being.
Key elements of oral homeostasis
Oral mucosa
The specific structural and functional organization of the epithelial lining of the oral cavity
serves a key role in oral health maintenance. The oral epithelium provides a physical
barrier to the outside world. A break in this barrier can easily lead to invasion of harmful
agents into the body and particularly the exposure of the immune system to various
microorganisms. Additionally, the human oral mucosa may be considered a first line of
defence against invading pathogens as the oral cavity is a site where many antigens are
initially encountered by the body. The role of epithelial cells has been proposed as an early
warning system or sensor for infection (Eckmann et al., 2000; Aldridge et al., 2005).
The oral mucosa is anatomically divided into three tissue layers: 1) epithelium; 2)
basement membrane, and 3) connective tissue. The epithelium consists of approximately
40-50 layers of stratified squamous epithelial cells. Related to its many functions, the oral
cavity contains several different types of stratified squamous epithelia, including those
classified as nonkeratinized, parakeratinized, and orthokeratinized (Brukhardt and
Maerker, 1981). Primarily nonkeratinized epithelium provides a lining in the cheeks, lips,
floor of mouth, ventral aspect of the tongue, soft palate, and upper and lower vestibular
sulci. Parakeratinized and orthokeratinized epithelium lines the hard palate and the mucosa
surrounding the teeth (Grafstrm, 2002). The major oral cell types are keratinocytes and
12
gingival epithelial cells (Krisanaprakornkit et al., 2000) that express specific pattern of
cytokines/chemokines that distinguish them from the epithelium in the gastrointestinal
tract (Formanek et al., 1999). In addition to the innate barrier function they perform,
gingival epithelial cells are also capable of expressing two anti-microbial peptides of the
-defensin family, human -defensin 1 and human -defensin 2. The role of -defensins
has been defined in innate host defense against various oral microorganisms
(Krisanaprakornkit et al., 2000, Devine, 2003, Eberhard et al., 2009; Gardner et al., 2009).
Tissues of the oral cavity are constantly exposed to innate defenses derived from saliva,
gingival crevicular fluid (GCF), epithelial cells, and neutrophils, and host-defense peptides
(HDPs) are significant in all of these, working in synergy with other defense components.
Saliva
Saliva effectively mediates the fine coordination of various functions of the oral cavity
and plays an important role in the maintenance of the overall health. Whole saliva is a
complex mixture of parotid, submandibular, sublingual and minor salivary gland
secretions mixed with bacteria, leukocytes, desquamated epithelial cells, and crevicular
fluid (Tenovuo, 1989). Saliva is a multifunctional secretion containing components that
contribute to oral buffering, lubrication, enamel mineralization, taste, digestion and
aggregation (e.g. agglutinins and mucin, MUC5B and MUC7) (Nieuw Amerongen and
Veerman, 2002). In addition to its flushing and clearing effect saliva with its intricate
composition provides reliable defense against external irritants and contributes to the
maintenance of the integrity of oral homeostasis. Constituents that are either directly
antimicrobial or interfere with microbial colonization or nutrition include, for example,
HDPs, secretory IgA, lactoferrin, lysozyme, sialoperoxidase, myeloperoxidase, chitinase,
calprotectin, and chromagranin A (Schupbach et al., 2001; Vitorino et al., 2005; Shimada
2006).
Oral microbiota
Along with its fine structural organization the oral cavity is unique with its specific
microbiota comprising an astonishing variety of species residing in oral biofilms as well as
in a planktonic state in the oral fluids. The predominant genera detected in the oral cavity
include Streptococcus, Actinomyces, Veillonella, Fusobacterium, Porphyromonas,
Prevotella, Treponema, Neisseria, Haemophilus, Eubacterium, Lactobacillus,
Bifidobacterium, Capnocytophaga, Capnocytophaga, Peptostreptococcus, Staphilococcus,
and Propionibacterium (Wilson, 2005). Resident commensal populations protect tissues
from colonization by exogenous pathogens, promote normal development of host cell
structure and function, ensure normal development of the immune system, and coordinate
immune responses (Devine and Cosseau, 2008). More than 1000 bacterial species have
been identified from the human mouth (Keijser et al., 2008; Paster et al., 2006), and only
50-60% of these microorganisms can currently be cultured. A plausible explanation for
13
this intricacy is that some species have evolved to live within a biofilm community of
interdependent species and cannot grow in monoculture (Wade, 2002; Handelsman, 2004).
An investigation of the bacterial flora found in healthy volunteers showed that a given
individual is colonized by 30 to 80 of the possible 1000 species at any given time (Aas et
al., 2005). Within biofilms, resident bacteria gain significant advantages, that is, protection
of host defenses and antimicrobial agents; expression of resistant phenotypes; and the
development of food-webs and interactions such as quorum-sensing (Marsh, 2005;
Roberts and Mullany, 2006; Bamford et al., 2009; Hojo et al., 2009; Keller and Costerton,
2009). The beneficial role of commensal microbiota has been evaluated in various in vitro
settings indicating that some microbes can suppress epithelial cell cytokine responses
(Hasegawa et al., 2007; Cosseau et al., 2008); determine normal expression of immune
mediators (Dixon et al., 2004); and provide protection against colonization by exogenous
microorganisms (Marsh, 2005). In general, microbial populations of the mouth are
numerous, diverse and site-specific.
The oral microbiota plays critical roles in human health and is directly linked to
diseases such as dental caries and periodontal diseases.
2.2 Common oral diseases

Dental caries and periodontal disease are the most common bacterial diseases of man
which result from an interaction between a susceptible host, commensal microbiota and
the environment. Although some specific microorganisms have been implicated in the
pathogenesis of these conditions, it is now recognized that they are not classical infectious
diseases but rather a complex of diseases resulting from a breakdown in the homeostasis
between the human host and microbiota.
2.2.1 Dental caries

Dental caries remains one of the principal diseases in the oral cavity with a significant
social impact. Caries is a result of the complex interaction between carbohydrates in food
and cariogenic microorganisms in oral biofilms, influenced by the quality and quantity of
saliva, and clinically manifested by demineralization and destruction of dental hard
tissues. Recent development in molecular analyses have shown that all the bacteria that
have been associated with caries belong to the normal microbiota of the oral cavity and
dental caries is regarded as an endogenous infection (Fejerskov and Nyvad, 2003;
Takahashi and Nyvad, 2008). Three major hypotheses for the etiology of caries have been
supported: the specific plaque hypothesis, the non-specific plaque hypothesis, and the
ecological plaque hypothesis (Loesche 1992; Marsh 1994; Martin et al., 2002). In light of
14
the ecological plaque hypothesis caries is a result of a shift in the balance of resident
microbiota driven by changes in local environmental conditions (Aas et al., 2008). It is
generally believed that all three parameters (microorganisms, the host, and environment)
must act simultaneously for carious lesions to develop and progress and to become
visually detectable (Shaw et al., 2008).
A wide group of microorganisms are identified from carious lesions of which
Streptococcus mutans, Lactobacillus acidophilus, and Actinomyces viscosus may be
considered the main pathogenic species involved in the initiation and development of
dental caries (Shivakumar et al., 2009). Streptococcus mutans, initially isolated in 1924,
has been primarily implicated in this disease (Hamada and Slade, 1980; Loesche, 1986)
and extensively studied throughout several decades. Some significant virulent traits of S.
mutans that contribute to caries initiation and progression are: (i) initiation of biofilm
formation by adherence and accumulation on the tooth surface that is promoted by its
synthesis of insoluble, extracellular polysaccharides; (ii) production of numerous
bacteriocins that kill other species, favouring its competition in dental biofilms; (iii) high
efficiency in catabolizing carbohydrates and producing acids; and (iv) the ability to
tolerate low pH (Belli and Marquis, 1991; Li and Burne, 2001; Kuramitsu, 2003; Scheie
and Petersen, 2004). Various studies have shown that the expression of virulence traits by
S. mutans requires multiple signal transduction pathways and complex regulatory
networks. A signal peptide-mediated quorum-sensing system encoded by comCDE genes
has been found to play a central role in regulation of genetic competence, bacteriocin
production, biofilm formation and stress response (Li et al., 2001a, b, 2002a; van der
Ploeg, 2005). Additionally, the genes that appear to be important for the cariogenicity of S.
mutans, are regulated at transcription level (Jayaraman et al., 1997; Hiratsuka et al., 1998).
Although numerous in vitro studies provide evidence of molecular mechanisms of S.
mutans cariogenicity and this species appears the most extensively studied, in vivo test
models do not generally validate basic laboratory findings. For example, Aas et al., (2008)
have demonstrated that 10% of subjects with rampant caries do not have measurable levels
of S. mutans. No detectable levels of S. mutans were also reported in 10 to 15 % of cariesactive subjects, thus indicating that the presence of S. mutans does not necessarily reflect
caries activity (Beighton 2005). Furthermore, phenotype of a bacterium expressed in
laboratory culture may not represent the properties expressed by the same organism in
vivo.
Key findings in the diversity of oral microbial species during past 10 years have
changed the view of the etiology of caries. Molecular biology techniques have shown that
more than 50% of the oral species are uncultivable by conventional methods (ten Cate
2009). It is now recognized that caries results not solely because of the presence of S.
mutans or any single organism in dental plaque, but it is rather the interaction of multiple
acid-producing organisms such as low-pH non-mutans streptococci, Veilonella,
Lactobacillus, Propionibacterium, Bifidobacterium that may be involved in the initiation
of the disease (Aas et al., 2008; He et al., 2009; Matzourani et al., 2009). The ecological
plaque hypothesis suggests that the cariogenic oral environment will select for increased
proportions and numbers of acidogenic and aciduric microbiota with certain taxa
exhibiting a reduced presence under these conditions (Matzourani et al., 2009).
15

The classical treatment plan for caries yet remains to be the operative approach of
complete caries removal. A series of novel methods of caries removal have been
described; including chemomechanical caries preparation, air abrasion, sono-abrasion,
polymer rotary burs and lasers (Ricketts and Pitts, 2009). However, more scientific efforts
are directed towards discovering effective methods for caries prophylaxis based on
inhibiting the known mechanisms of caries development. The elimination of cariogenic
bacteria from the oral cavity using antibacterial agents is one of the primary strategies for
the prevention (Wicht et al., 2003; Caufiled, 2005; Altman et al., 2006; Modesto and
Drake, 2006; Johansson et al., 2008). Fluoride treatment used worldwide has successfully
limited caries progression, but was not sufficient to control this infectious disease even
when used together with professional tooth cleaning and dietary counselling in
populations exposed to cariogenic microbiota (Haugejorden and Birkeland, 2005; Yee et
al., 2006; Akers, 2008; Carvalho et al., 2009). Polyphenols from plant stimulant beverages
like cocoa, coffee, and tea have shown pronounced antimicrobial effect against S. mutans,
and can additionally be implemented in the prevention of pathogenesis of dental caries
(Ferrazzano et al., 2009). Polyphenols in stimulant beverages significantly reduce biofilm
formation and acid production by S. mutans and S. sanguinis. Further, as an example, in
vitro studies have shown that S. mutans is susceptible to methanol and aqueous extracts of
Garcinia kola, Hibiscus sabdariffa (Afolabi et al., 2008).
Sugar substitutes have a long history of being effective in caries reduction. The main
sugar substitutes used are sorbitol and xylitol. Xylitol is not fermented by oral bacteria and
is considered to be non-cariogenic while sorbitol in solution can be fermented slowly by
mutant streptococci. Chewing sorbitol-sweetened gum does not cause a fall in plaque pH,
however (Edgar, 1998). A regular consumption of xylitol lozenges can modify dental
plaque resulting in marked reduction in the plaque acidogenicity (Splieth et al., 2009).
Active and passive immunization strategies which target key elements in the molecular
pathogenesis of mutans streptococci hold promise. Considerable caries reduction could be
attained if colonization of S. mutans could be prevented or reduced at the time of eruption
of both deciduous and permanent teeth. Thus, a successful vaccination directed against S.
mutans could be a valuable adjunct to other caries-preventive measures. However, S.
mutans being the sole target species in caries prophylaxis does not comply with the key
principles of ecological plaque hypothesis.
Bacteriotherapy further emerges as a fascinating approach in oral infectious disease
management. A daily application of JH145, a naturally occurring LDH-deficient variant of
S. rattus, could compete with S. mutans for its habitat on the tooth surface and thus
contribute to caries prevention (Hillman et al., 2009).
16
2.2.2 Periodontal disease

Periodontal disease has been described as a heterogeneous group of pathoses
characterized by a predominance of specific infectious agents in the face of inadequate
local host defenses (Slots, 2005). The definition reflects the complexity of periodontal
disease.
A primary risk factor considered in the etiology and progression of periodontal disease
is the infection by specific bacterial pathogens. The actions of bacterial virulence factors,
directly or indirectly through the activation of the immune system, cause swelling,
inflammation, and gingival pocket formation. The balance between protective and
destructive immune responses is a key determinant of disease progression. This balance is
strongly influenced by the host response to the challenge caused by subgingival bacteria
(Sakamoto et al., 2005; Teng, 2006a, Teng, 2006b). Socransky et al., (1998) have
formulated a color coded complex for periodontal pathogens with respect to their
destructive potential. The Red complex, which includes Porphyromonas gingivalis,
Treponema denticola, and Tannerella forsythia, strongly correlated to chronic periodontal
disease, and the first two species together with Aggregatibacter actinomycetemcomitans
are currently recognized as the main causative species of periodontal disease (Borrell and
Papapanou, 2005; Nrskov- Lauritsen and Kilian, 2006).
Destruction of the periodontal ligament and resorption of the alveolar bone leading to
tooth loss is the hallmark of periodontal disease. Since host and microbiota interactions
are dynamic, disease may arise at the mucosal surface of a susceptible host when a
perturbation occurs in the epithelial environment, for example, when the host becomes
immunocompromised, or as a result of the unintended (in an evolutionary sense)
consequences of bacterial activity (Galan and Zhou, 2000). The initial interface between
the host and the potentially periodontopathic organisms, such as P. gingivalis and A.
actinomycetemcomitans, is the epithelial layer that lines the subgingival crevice. Epithelial
cells are both a physical barrier to infection and a component of a network that efficiently
signals microbial intrusion to the immune cells to insure effective mobilization of the
innate and specific defense mechanisms (Kagnoff and Eckmann, 1997). Studies have
shown that several periodontal pathogens, A. actinomycetemcomitans, P. gingivalis, F.
nucleatum, and T. denticola, can effectively invade and exist in oral epithelial cells
(Sreenivasan et al., 1993; Rudney et al., 2005; Vitkov et al., 2005; Sakakibara et al.,
2007). Furthermore, intracellular P. gingivalis is able to inhibit apoptosis, a feature that
may contribute to bacterial persistence and chronic, slowly progressing tissue destruction
(Nakhjiri et al., 2001). Localization of bacteria in host tissues provides an ideal position
from which the microorganism can effectively deliver toxic molecules and enzymes and at
the same time can avoid host defense mechanisms. For example, P. gingivalis is able to
inhibit production of IL-8 by epithelial cells, which may provide the microorganism with
an advantage in evading polymorphonuclear(PMN)-mediated killing (Dareveau et al.,
1998).
17
Host tissue damage can be due to bacterial properties resulting directly in degradation
of host tissues and those causing release of biologic mediators from host tissue cells that
lead to tissue destruction. A large group of enzymes produced by periodontal
microorganisms appear capable of degrading host tissues and intercellular matrix
molecules. Bacterial products may perturb the immune system resulting in tissue
destruction. The proportion of P. gingivalis, A. actinomycetemcomitans, T. forsythia and
the number of CD4(+) T are higher in active than in inactive sites (Silva et al., 2008).
Pathologically increased activity of matrix metalloproteinases (MMP-2; MMP-8;
MMP-9; MMP-13) in inflamed periodontal structures leads to periodontal destruction due
to collagen degradation (Biyiko lu et al., 2009; Gu et al., 2009; Marcaccini et al., 2009;
Yamazaki-Kubota et al., 2009). Furthermore, some periodontal pathogens may indirectly
contribute to tissue damage by induction of host tissue proteinases such as elastase and
MMPs (Pattamapun et al., 2003; Tiranathanagul et al., 2004; Bodet et al., 2007; Guam et
al., 2008). A. actinomycetemcomitans and P.gingivalis can elevate MMP-2 secretion in
human periodontal ligament fibroblasts (PDLFs), indicating that periodontal pathogens
play an important role in tissue destruction and disintegration of extracellular matrix in
periodontal diseases (Chang et al., 2002).

Traditionally periodontal disease treatment is a four-phase approach including nonsurgical periodontal therapy, surgical procedures, restorative treatment, and supportive
care or maintenance. The foremost goal in periodontal therapy is the elimination or
reduction of the pathogenic potential of dental plaque. However, even with appropriate
treatment and improved oral hygiene many patients fail to respond to therapy unless
certain factors (e.g. smoking, uncontrolled diabetes) are also eliminated.
One strategy to prevent periodontal disease may be the controlling of factors that
disrupt the microbial ecological balance from a symbiotic and healthy to a host-pathogen
relationship which then leads to disease (Kinane et al., 1999). Systemic and local
antibiotic applications have been used as adjunct to conventional periodontal therapy.
However, because of the chronic nature of periodontitis antibiotic medications are not
generally used except in patients who do not respond to conventional therapy.
Inappropriate use of antibiotic agents can lead to overgrowth of potentially pathogenic
organisms and development of bacterial drug resistance.
A novel prophylactic strategy in periodontal disease management that merits further
investigations is the replacement of common periodontal pathogens by commensal oral
microbes. Teughels and coworkers have tested the hypothesis that the subgingival
application of S. sanguinis KTH-4, S. salivarius TOVE and S. mitis BMS after mechanical
debridement would enhance the microbial shift away from periodontopathogens (Teughels
et al., 2007). A significant delay in recolonization of periodontal pockets by A.
actinomycetemcomitans, P. gingivalis, P. intermedia, and Tannerella forsythia after root
planing was observed when the above species were locally applied.
18
Scientific understanding of molecular mechanisms in the development and progression

of common oral diseases can foster the implementation of natural host defense
mechanisms to combat oral infections. The application of health-promoting bacteria for
therapeutic purposes is one interesting field in this regard.
2.3 Probiotics from the oral health perspective
2.3.1 Probiotics
2.3.1.1 Emergence and definition of the term

The word probiotic is derived from the Greek probiosis meaning for life and generally
applies to bacteria causing no harm to the host. The probiotic concept dates back over 100
years, and associates with the name of the Ukranian bacteriologist and Nobel Laureate Ilie
Metchnikoff, who proposed the scientific rationale for the beneficial effects of lactic acid
bacteria. In 1888 while working in the Pasteur Institute in Paris, Ilie Metchnikoff
emphasized a theory that putrefactive-type fermentation products by some gut pathogens
may be the cause for autointoxication of the macroorganism. Furthermore, the intake of
bacteria involved in yogurt fermentation, L. bulgaricus and Streptococcus thermophilus,
can effectively suppress metabolic activity of pathogenic intestinal species thus
maintaining health. He claimed that the longevity of some populations in Bulgaria, Turkey
and Armenia was due to regular consumption of fermented milk products rich in live lactic
acid bacteria (LAB). The scientist has promoted the idea that LAB in yogurt may
neutralize deleterious effects of gut pathogens, thus extending life span. He further
contributed to the adoption of the name of the species, L. bulgaricus, one of the essential
yogurt starter microorganisms. This also meant the birth of modern dairy industry
(Meurman, 2005).
Despite inconclusive evidence of health effect of yogurt bacteria research interest
intensified in the later years. Ferdinand Vergin was the first (1954) to introduce the term
probiotic mainly opposing it to antibiotics. Kollath (1954) used the term to designate
active substances that are essential for healthy development of life. Lilly and Stillwell
(1965) contributed to the adoption of probiotics as scientific term providing evidence that
bacteria secrete substances that stimulate the growth of another. The definition underwent
further modifications broadening its meaning. Parker (1974) defined them as: organisms
and substances which contribute to intestinal balance. The closest to contemporary
meaning of probiotics has been given by Roy Fuller (1989): a live microbial feed
supplement which beneficially affects the host animal by improving the intestinal
microbial balance. Yet probiotic activity was limited to live bacterial species. However,
accumulation of new research data contributed to the understanding of probiotic activities
beyond the scope of gastrointestinal tract and bacterial cell fractions were also claimed
19
effective. Although the concept of probiotics remains open to further modifications, in

2002 the Joint Food and Agriculture Organization/World Health Organization Working
Group (FAO/WHO) officially formulated the term probiotics: Live microorganisms
which when administered in adequate amounts confer a health benefit on the host. This
definition was adopted by the International Scientific Association for Probiotics and
Prebiotics (Reid et al., 2003). The definition retains the historical elements of the use of
living organisms for health purposes but does not restrict the application of the term only
to oral probiotics with intestinal outcomes.
Probiotics can also target the oral cavity, nasopharynx, stomach, vagina, bladder and
skin. Another implication of the FAO/WHO definition is that unless strains are shown to
confer clinically established physiological benefits, they should not be referred to as
probiotics (Reid et al., 2003). Under the formulation of the latter definition probiotics are
linked to food and to food only, thus excluding any reference to the term biotherapeutic
agents. However, emerging data suggest that while viable organisms may be most
effective for specific effects, non-viable probiotic organisms (abiotics) may be efficacious
in specific situations (Salminen et al., 1999; Shortt, 1999). It is likely that the abiotic idea,
if accepted, will further broaden the health potential of the probiotic concept in the future
(Shortt, 1999).
The principal microorganisms in use as probiotics belong to the genera Lactobacillus
and Bifidobacterium. However, other genera including Escherichia, Enterococcus, and
Saccharomyces are also used. Lactobacilli and bifidobacteria constitute the two most
important probiotic groups under consideration owing to their recognition as members of
the indigenous microbiota of humans, their history of safety and the general body of
evidence that supports their positive roles. At this stage, phylogenetics has recognized 97
species of lactobacilli (Dellaglio and Felis, 2005), 18 of which are considered to be of
some interest in probiotics; and 31 species of Bifidobacterium, 11 of which have been
detected in human feces (Sanders, 1999). Lactic acid bacteria are associated with habitats
that are rich in nutrients, such as various food products. They can be found in soil, water,
sewage, and they can ferment or spoil food. Certain LAB species are inhabitants of the
human oral cavity, the intestinal tract, and the vagina, and may have a beneficial influence
on these human ecosystems (Holzapfel et al., 2001).
2.3.1.2 Beneficial effects of probiotics

Numerous health benefits have been proposed to result from consumption of probiotic
bacteria. Although the specific mechanisms involved in the many suggested benefits have
not been completely established, evidence suggests that probiotics can influence various
disease conditions in a positive manner. Table 1 outlines the most common clinical
conditions with a positive outcome after probiotic administration.
20
Table 1. Clinical conditions improved by probiotic intake
Disorder
Probiotic
Patient Duration
Clinical effect
Reference
12
Induction of
Rembacken et
months
remission;
al., 1997
group
GI disorder
Ulcerative
E. coli Nissle 1917
116
colitis
prevention of
relapses
E. coli Nissle 1917
B. longum
120
120
12 weeks
4 weeks
Maintaining the
Kruis et al.,
remission
2004
Improved
Fujimori et al.,
systemic
2009
function
VSL#3
L. rhamnosus GG
29
187
12
Remission
Miele et al.,
months
maintenance
2009
12
Prolongation of
Zocco et al.,
months
relapse-free
2006
time
E. coli Nissle 1917
Saccharomyces
327
25
12
Induction of
Kruis et al.,
months
remission
2001
4 weeks
Induction of
Guslandi et al.,
remission
2003
Improved
Garcia Vilela et
intestinal
al., 2008
boulardii
Crohn`s
Saccharomyces
disease
boulardii
34
3 months
permeability
L. johnsonii
98
6 months
Postsurgical
Marteau et al.,
Crohn`s disease
2006
recurrence
E. coli Nissle 1917
Genetically
24
10
3 months
7 days
21
Relapse rate
Guslandi et al.,
decreased
2000
Decrease in
Braat et al.,
disease activity
2006
12
Maintaining the
Mimura et al.,
months
remission
2004
4 weeks
Prolongation of
Gionchetti et
remission
al., 2007
On
Decreased
Montes et al.,
intake
symptoms of
1995
modified L. lactis
(LL Thy12)
delivering IL-10
Pouchitis
VSL#3
VSL#3
Lactose
L. acidophilus
36
23
20
maldigestion
lactosemaldigestion
Diarrhea
L. rhamnsosus GG
204
episodes
15
Reduction of
Oberhelman et
months
diarrhea
al., 1999
episodes in
children
L. rhamnosus
69
5 days
19070-2; L.
Reduction of
Rosenfeldt et
diarrhea phase
al., 2002
Improved
Sarker et al.,
management of
2005
reuteri DSM
12246
L. paracasei ST 11
230
5 days
non-rotavirus
diarrhea
L.rhamnosus GG
140
5 days
Shorten
Guandalini et
diarrhea
al., 2000
duration
Probiotic
75
5 days
combination
Allergy states
L. acidophilus
47
4 months
NCFM; B. lactis
Shorten
Teran et al.,
diarrhea periods
2009
Prevention of
Ouwehand et
pollen-induced
al., 2009
infiltration of
eosinophils
22
Lactobacillus F19
89
7 months
Prevents early
West et al.,
manifestation of 2009
allergy
L.GG; L.gasseri
40
10 weeks
TMC0365
Decreased
Kawase et al.,
allergic rhinitis
2009
symptoms
Mechanisms of action explaining the beneficial probiotic effects, though still unclear, may
include the modulation of host immune response leading to strengthening of the resistance
to pathogenic challenge; alteration of the composition and metabolic activity of host
microbiota at the specific location; interference with pathogen adhesion and growth
inhibition (Hatakka and Saxelin, 2008).
2.3.1.3 Selection criteria for probiotic candidates

A wide range of requirements have been discussed as related to various applications of
probiotics (Lee, 2009), but among key selection criteria with emphasis on human health
are:
Adhesion and colonization (at least transitory) of human body. Adhesion may
increase the retention time of a probiotic and place bacteria and host surfaces
(body fluids and epithelial cells) in close contact thus facilitating further
probiotic activity;
Enhancement of the non-specific and specific immune response of the host;
Production of antimicrobial substances and competition with pathogens for
binding sites;
Survival and resistance to human defense mechanisms during the oro-gastrointestinal transit;
Human safety.
Additionally, the probiotic candidate should (1) be of human origin; (2) be non
pathogenic; (3) confer clinically established physiological benefits; and (4) maintain
viability and activity throughout product manufacture and processing (Dunne et al., 1999;
Reid et al., 2003). Generally the list of criteria for probiotic selection is application-based
and depends on the specific probiotic effects desired and the target site of action.
Oral cavity with its complex anatomy with both soft and hard tissues and functional
integrity is a new area for studies of probiotic therapy in the treatment and prevention of
most common disorders in the mouth. Species investigated from an oral health perspective
are given in Table 2.
23
Table 2. Probiotic candidates for the oral cavity
Lactobacillus
Bifidobacterium Streptococcus
L. reuteri
B. lactis
S.
Propionibacterium Weissella
salivarius P. freudenreichii
W. cibaria
K12
L. plantarum
B. longum
L. rhamnosus
B. infantis
S. thermophilus
L. salivarius
L. acidophilus
L. casei
L. johnsonii
Among the selection criteria with relevance of probiotics to the mouth are:
Attachment, adhesion, and oral colonization;
Resistance to oral defence mechanisms;
Production of antimicrobial substances and competition with pathogens for
binding sites;
Carbohydrate and protein utilization patterns;
Enhancement of the non-specific and specific immune response of the host;
Safety to oral ecology and oral structures.
2.3.1.3.1 Attachment, adhesion, and oral colonization

The evidence is scarce regarding the question whether probiotics permanently reside in the
human body and in the mouth, in particular (Petti et al., 2001; Yli-Knuuttila et al., 2006).
However, it can be anticipated that among the 103 104 CFU/g lactobacilli found in the
oral cavity (Bernardeau et al., 2008) there are species/strains capable of exerting probiotic
properties. Bacteria reside in the mouth either in planktonic state or are finely integrated in
biofilm on various oral surfaces. Oral biofilms are dynamically changing and develop
increasingly complex structures as they mature. Interaction between species is
characteristic in biofilms. Some species may depend on others to provide favorite
environment for colonization. Furthermore, bacteria in biofilms differ physiologically
from their planktonic counterparts and tend to be much more resistant to environmental
factors and antimicrobial agents. It has been established that distinct genes become active
when planktonic bacteria bind to surfaces and grow in biofilms (Burne et al., 1999;
Rudney, 2000). On the other hand, saliva is the essential medium in the mouth
contributing to the microbial diversity. It plays an integral role in propagating oral
biofilms. Salivary flow can easily lead to detachment of some microbes from biofilm
24
surfaces and thus modulate microbial colonization. Furthermore, as complex medium

saliva contains different proteins with bactericidal, bacteriostatic, or inhibitory activity that
collectively may affect a variety of species in planktonic state (Germaine and Tellefson,
1986; Rudney et al., 1991; Hahnel et al., 2008; Grlsch et al., 2009). Biofilm species
composition can also depend on phenomena like auto- or co-aggregation that may prevent
microorganisms from establishing themselves in the biofilms. Hence by taking into
consideration the multifaceted nature of biofilm development and multivariate species
interactions we can acquire better understanding and interpretation of studies with
probiotics in the oral cavity.
There are very few studies of the colonization of probiotic bacteria in the oral cavity,
and the results are contradictory. The pattern of oral colonization by probiotic species has
been found to be transient and gradually diminished soon after probiotic administration
period ended (Bussscher et al., 1999; Petti et al., 2001; Yli-Knuuttila et al., 2006).
Therefore, it is necessary to consider that the associations and mechanisms less intricate
and more transient than those of native microflora may mediate probiotic effects. On the
other hand probiotic administration early in life may provide those species the opportunity
to interact with host receptors early and subsequently integrate in microbial communities
resulting in permanent condition. In a study comparing species variability in the mouth
and feces Ahrne et al. (1998) have discovered that species most frequently recovered from
the rectal as well as from the oral mucosa were L. plantarum and L. rhamnosus, which
were present in 52% and 26% of the individuals, respectively. However, this study did not
define those strains as permanent colonizers of the two sites tested or whether the mouth is
their natural habitat. The most common species of lactobacilli recovered from saliva of a
Thai population were L. fermentum and L. rhamnsous (Teanpaisan et al., 2006). A
promising finding was that lactobacilli population differed between healthy individuals
and those with periodontal disease. Koll-Klais et al. (2005) have observed that healthy
persons are populated by L. gasseri and L. fermentum, whereas the predominant species in
periodontitis patients was L. plantarum while the first two were undetectable.
Observations by this study group showed that microorganisms with probiotic properties
may indeed exist and reside in the oral cavity. However, the complexity of biofilm
development and interspecies interactions require more thorough investigations in order to
assert true probiotic candidates with activity in the oral cavity.
The mechanism of adhesion to oral surfaces is an issue of importance for the long term
probiotic effect. The capacity of probiotics to adhere to surfaces of the oral cavity can
avoid or at least reduce rapid clearance from the environment. Among the different assays
available to study the adhesion phenomenon, two model systems predominate: systems
using saliva-coated hydroxylapatite (HA) and hydroxylapatite coated with buffers,
proteins, and other substances (Ostengo and Nader-Macias, 2004). Probiotics and putative
probiotic strains have been shown to vary extensively in their adhesiveness to salivacoated surfaces. Lactobacilli have shown better adherence than bifidobacteria to salivacoated hydroxyapatite beads and polystyrene plates (Haukioja et al., 2006). Furthermore,
interplay between saliva and probiotics may additionally modify composition of salivary
pellicle thus altering the attachment pattern. In vitro removal of a heavy molecular weight
protein band that contained salivary agglutinin gp340 has been observed after incubation
25
of saliva with four commercially available probiotic strains (Haukioja et al., 2008). The
mechanisms of adhesion of lactobacilli involve hydrophobicity and surface charge, as well
as specific carbohydrate and/or proteinaceous components (Lorca et al., 2002). The
interaction between bacterial and HA surfaces have been shown to depend not only on the
nature and number of available anchoring groups, but also on the calcium ions in the
medium that bind the functional groups of the bacteria to the biomaterial (Venegas et al.,
2006).
The adhesion of probiotic bacteria to oral soft tissues is another aspect that promotes
their health effect to the host. Cell adhesion is a complex process involving contact
between the bacterial cell and interacting surfaces. Secretome studies can provide valuable
information about bacterial structures responsible for binding to host surfaces. The domain
composition of the L. plantarum proteins predicted appeared to be involved in the
adherence to extracellular macromolecules (Boekhorst et al., 2006).
2.3.1.3.2 Resistance to oral defence mechanisms

It is generally considered that to be able to exert its beneficial effect the probiotic
candidate should survive the oro-gastrointestinal passage. Ingested probiotics are exposed
first to saliva which mediates the contact with hard and soft oral tissues. During this first
step of contact with tissues resistance to environmental factors in the mouth are of
paramount importance for bacteria to survive. Salivary proteins such as lysozyme,
lactoferrin, histatin, salivary peroxidase, cystatins, and secretory IgA can collectively
affect viability or cell surface morphology of probiotic species and further affect their
adhesion and metabolic activity. Saliva can kill or damage species in planktonic state as
well as mediate intra- and interspecies aggregation, thus additionally affecting adhesion.
The role of saliva on microbial establishment can be contradictory, however, inhibiting
colonization on one hand (by growth inhibition, killing, or prevention of adherence to host
tissues), and promoting microbial colonization, on the other hand (Bosch et al., 2003). In
vitro studies testing probiotic survival in saliva have shown that Lactobacillus and
Bifidobacterium strains cannot grow in saliva but remain viable after 24 hours of
incubation (Haukioja et al., 2006). Lysozyme pretreatment has been observed to
significantly reduce the adhesion of L. rhamnosus GG, L. rhamnosus Lc705 and L. casei
Shirota. However, the adhesive properties of L. johnsonii La1 and B. lactis Bb12 remained
unaffected. These results emphasize the strain-specific response to proteolytic enzymes
and this feature needs to be considered when selecting probiotics for the oral cavity.
2.3.2 Clinical relevance of probiotics in the oral cavity

Probiotic relevance in the management of common oral diseases has been advocated in a
number of clinical studies. Table 3 lists the species/strains that have been observed to
positively affect infectious oral diseases.
26
Table 3. Clincal trials with positive effect after probiotic administration
Oral disease
Probiotic
Vehicle of
Duration
administration
of the
Result
Reference
study
Dental
L. rhamnosus
caries
GG
L. rhamnosus
Cheese
3 weeks
Milk
7 months
GG
Reduction of
Ahola et
S. mutans
al., 2002
Lower S.
Nse et
mutans
al., 2001
counts
Bifidobacterium
Yogurt
4 weeks
DN-173010
B. animalis
Fruit yogurt
4 weeks
subsp. lactis
Reduction of
aglar et
S. mutans
al., 2005
Reduction of
Cildir et
S. mutans
al., 2009
Improved
Krasse et
gingival
al., 2006
DN-173010
Gingivitis
L. reuteri
Chewing
2 weeks
tablet
and
health and
periodontitis
reduced
plaque
accumulation
L. reuteri
Chewing gum
2 weeks
Improved
Twetman
bleeding on
et al.,
probing, and
2009
decrease of
GCF volume
L. casei 37
Periodontal
Several
Reduction of
Volozhin
dressing
days in
periodontal
et al.,
periodontal pathogens
2004
dressing
C. albicans L. rhamnosus
infections
Cheese
16 weeks
GG; P.
27
Decreased
Hatakka
prevalence
et al.,
freudenreichii
of
ssp.shermanii
C.albicans
2006
JS
2.3.2.1 Probiotics and dental caries

The first randomized, double-blind, placebo-controlled intervention study of L. rhamnosus
GG effect on dental caries was completed in 2001; the study included 594 children, 1-6
years old, who consumed milk for 7 months (Nse et al., 2001). Probiotic milk was able to
reduce S. mutans counts at the end of the trial and a significant reduction of caries risk was
also observed. S. mutans reduction was also achieved after consumption of L. rhamnosus
LC 705, Bifidobacterium DN-173 010, and B. lactis Bb-12 in cheese, yogurt, or ice-cream
as vehicles (Ahola et al., 2002; aglar et al., 2005; aglar et al., 2008). Fermented dairy
products being a favorable habitat for lactobacilli are generally used as vehicles for
probiotic administration. However, for the scope of the oral cavity several other means of
administration have been assessed. A tablet, a telescopic straw, a lozenge, and a chewing
gum containing probiotics have shown reduction of common caries pathogen after 3-week
regular intake (aglar et al., 2006; aglar et al., 2007; aglar et al., 2008). The observed
positive correlation between probiotic intake and reduction in caries pathogen counts
might be a useful strategy in caries prophylaxis for some special risk groups. For example,
orthodontic patients wearing fixed appliances can experience higher caries risk during
treatment. Cildir et al. (2009) have shown that a probiotic intake of B. animalis subsp.
lactis DN-173010 can positively reduce salivary mutans streptococci in orthodontic
patients.
Possible explanation for the clinical results of probiotic intake may be the competition
for binding sites in oral biofilms as shown in some in vitro studies (Haukioja et al., 2008).
However, this area also calls for more in-depth studies.
2.3.2.2 Probiotics and periodontal disease

Only few clinical studies outlining probiotic effectiveness in periodontal disease have
been published to date. From the periodontal health perspective it should be noted that the
composition of lactobacilli species differs in healthy and periodontitis patients and that
obligately homofermentatives are less prevalent in chronic periodontitis (Koll-Klais et al.,
2005). A fourteen-day intake of L. reuteri led to the establishment of the strain in the oral
cavity and significant reduction of gingivitis and plaque in patients with moderate to
severe gingivitis (Krasse et al., 2006). Salivary inflammatory markers of periodontal
disease can be positively affected in smokers after L. salivarius WB21 tablet form
administration for eight weeks (Shimauchi et al., 2008). Periodontal inflammation has
been reduced after the intake of probiotic tablets (Bifidumbacterin and Acilact) available
28
on the Russian market (Grudianov et al., 2002). Studies from Russia have also shown that
a periodontal dressing containing L. casei 37 can reduce the number of most common
periodontal pathogens and extend remission up to 10 12 months (Volozhin et al., 2004).
Possible explanation to the results might be the inhibitory effect of probiotics on pathogen
growth thus altering the composition of oral biofilm. Due to its ability to inhibit P.
gingivalis, L. salivarius TI 2711 was given for 4 or 8 weeks in a tablet to healthy
volunteers at a concentration of 2x107 CFU/ml. A significant reduction of blackpigmented
rods in saliva was observed (Ishikawa et al., 2003). Additional finding in this study was
the increase of pH to neutral after treatment, thus highlighting both caries and periodontoprophylactic properties. The effectiveness of the latter Lactobacillus strain has been
confirmed by Matsuoka et al., (2006).
A proposed mechanism of action of probiotics is strengthening the mucosal barrier via
tropic effects on the epithelium and stimulating both the innate and adaptive immune
response. A double-blind, placebo-controlled clinical trial with L. reuteri ATCC 55730
and ATCC 5289 taken in a chewing gum for 10 min twice daily has shown reduction of
pro-inflammatory cytokines TNF- and IL-8 in gingival crevicular fluid (Twetman et al.,
2009).
Because of the broad diversity of species residing in the mouth new probiotic
candidates may be anticipated to emerge adding to the array of already known strains. A
novel concept favoring periodontal health has been introduced by Teughels and coworkers (Teughels et al., 2007; Nackaerts et al., 2008) suggesting re-colonization of the
gingival pocket after scaling and root planning by species like S. sanguinis KTH-4, S.
salivarius TOVE and S. mitis BMS these strains then thought to be able to inhibit
adhesion of common periodontal pathogens. The foundation of the re-colonization concept
stands on the principle that subgingival application of oral streptococci would enhance the
microbial shift away from periodontopathogens.
2.3.2.3 Probiotics and other oral disorders

Among other oral conditions that may be favorably affected by probiotic administration
are Candida infections and halitosis.
Halitosis, foetor ex ore, has mainly been ascribed to the production of volatile sulfur
compounds (VSC) by Gram negative anaerobes residing in periodontal pockets and on the
tongue dorsum. Halitosis has been significantly improved in subjects after probiotic
intake. S. salivarius K12 taken in a lozenge after a mouth wash led to reduction of VSC
levels in 85% of the subjects (Burton et al., 2006). Furthermore, L. salivarius has been the
most prominent species detected in healthy subjects, whereas in individuals with halitosis
it was almost undetectable or only at very low levels (Kazor et al., 2003). W. cibaria is
another species with probiotic properties which has been shown to reduce VSC production
both in vitro and in vivo (Kang et al., 2006). A contributing factor to the malodor
reduction can be the ability of W. cibaria to co-aggregate with species known for their
VSC production (F. nucleatum, for example). The aggregation thus affects the source for
malodorous compounds in the oral cavity (Kang et al., 2005).
29
Candida albicans is the commonest pathogen of oral fungal infections. Probiotic

applications may alleviate symptoms and reduce pathogenic potential of Candida species.
A 16-week probiotic intervention study demonstrated a significant reduction by 75% of
high yeast counts in the elderly (Hatakka et al., 2007). The intake of L. rhamnosus GG
containing cheese associated with control of oral Candida also led to reduction of the risk
of hyposalivation as reported by the same authors. Although this is the only study
published on the role of probiotics on yeast infection in humans two other in vivo studies
on mice have shown that lactobacilli might indeed be effective in controlling oral
candidiasis. Elahi et al., (2005) have demonstrated a higher clearance of C. albicans in
mice fed with L. acidophilus compared to the control group. However, in another study no
noticeable delay in colonization of the oral cavity by C. albicans of immunocompromized
mice was achieved when heat killed L. casei and L. acidophilus cells were given (Wagner
et al., 2000).
2.4 L. delbrueckii subsp. bulgaricus as a probiotic

The discovery of Lactobacillus bulgaricus relates to Stamen Grigorov, a Bulgarian
microbiologist who in 1905, in the laboratory of Professor Masole in Geneva, isolated the
species from yogurt and thereafter the microorganism was named after the country.
Lactobacillus bulgaricus was formally described by Orla-Jensen in 1919 and validated
in 1971 with the study of Rogosa and Hansen (1971). After a number of different studies,
Weiss et al. (1984) proposed the union of Lactobacillus delbrueckii, Lactobacillus
leichmannii, Lactobacillus lactis and Lactobacillus bulgaricus under the name of L.
delbrueckii, and thereafter the name of the former Lactobacillus bulgaricus was
changed in Lactobacillus delbrueckii subsp. bulgaricus. Within the species, three
subspecies were recognized to exhibit DNA-DNA homologies of 90-100% among each
other (Howey et al., 1990; Torriani et al., 1997; Germond et al., 2003). Consequently, they
cannot be easily identified, not even by molecular methods, and can be mistakenly
confused if only the phenotypic characteristics are known (Milliere et al., 1996;
Vandamme et al., 1996; Giraffa et al., 2003).
L. delbrueckii subsp. bulgaricus is a Gram-positive, non-motile, obligatory
homofermentative, catalase-negative rod (Figure 1). Its DNA has 49-51 % GC ratio
(Hammes and Vogel, 1995), which is significantly higher compared to the GC content of
other lactobacilli in the genus (Nicolas et al., 2007). Carbohydrate fermentation results in
99.5 % D- and 0.5 % L-lactic acid. L. bulgaricus encodes many partial carbohydrate
metabolic pathways and shows a distinct preference for growth in lactose rich media. It
maintains extensive proteolytic and amino acid transport systems which are useful in the
protein-rich milk environment (Klaenhammer et al., 2008). L. delbrueckii subsp.
bulgaricus belongs to thermophylic lactic acid bacteria and temperatures between 43-46C
are optimal for its growth. Lactic acid bacteria can survive in anaerobic conditions because
oxygen is not needed for energy metabolism. They can tolerate aerobic environments as
well. A pH modified MRS (pH 4.58) agar and anaerobic incubation at 43C can be used to
30
selectively enumerate L. delbrueckii subsp. bulgaricus from a product (Tharmaraj and

Shah, 2003).
Figure 1. Scanning electron microscopy image of rod-shaped L. delbrueckii subsp. bulgaricus

(courtesy of Kari Lounatmaa).
Lactobacillus delbrueckii subsp. bulgaricus is one of the two bacteria required for the
production of yoghurt. It plays an essential role in the development of the organoleptic
(Ott et al., 1997; Petry et al., 2000), hygienic and perhaps probiotic properties of this food
(Hassan and Frank, 2001). Table 4 gives some of fermented milk products where L.
bulgaricus is used for production.
Table 4. Dairy products containing L. delbrueckii subsp. bulgaricus (source Lee and Wong, 1993).
Product
Starter microorganism
Yogurt
L. delbrueckii ssp. bulgaricus,

thermophilus
L. delbrueckii ssp. bulgaricus
Bulgarian butter milk

Dahi
Kefir
Kumys
Str.
L. delbrueckii ssp. bulgaricus, Str.

thermophilus, Leu. mesenteroides ssp.
cremoris
L. lactis ssp. lactis, L. lactis ssp. cremoris,
L. lactis diacetilactis, L. delbrueckii ssp.
bulgaricus,
Str.
thermophilus,
L.
helveticus, L. kefir, Saccharomyces ssp.
L. acidophilus, L. delbrueckii ssp.
bulgaricus, Saccharomyces lactis, Torula
koumiss
31
Yogurt has been considered the primary habitat of the species (Davis, 1975) because the
bacterium is highly adapted to milk environment (Norbert et al., 1983) and is also able to
resist low pH values (Delley and Germont, 2002). However, the millennium long tradition
of fermented milk production has propelled the search for plants serving as sources for L.
bulgaricus isolation. In a historical perspective plant extracts have been added to sheep
milk and then heated until a dense milk coagulum is obtained (Markoff, 1925). Several
plants have been reported as habitats for L. bulgaricus: Cornus mas, Ononis spinosa,
Berberis vulgaris, Paliurus aculeatus, Matricaria chamomilla, Prunus spinosa (Girginoff,
1959; Kantardjiev, 1962; Stefanova, 1985; Mychailova et al., 2007). Glucose, fructose,
mannose, and sucrose availability on leaf and stem surfaces of these plans are recognized
as nutritients that provide optimal growth conditions for the microbial species (Tukey,
1970; Schaffner and Beuchat, 1986; Andrews and Harris, 2000; Mercier and Lindow,
2000; Lee, 2001; Michaylova et al., 2005).
Relative debate exists about whether or not yogurt starter bacteria such as L.
bulgaricus should be considered probiotics. In vitro models and few clinical trials have
shown that yogurt bacteria cannot survive in the gastrointestinal tract thus being unable to
permanently colonize the gut (Shah and Jelen, 1990; Marteau et al., 1997; del Campo et
al., 2005; Garcia-Albiach et al., 2008). In contrast to the intestinal lactobacilli, L.
bulgaricus does not encode mucin-binding proteins and it is deficient of bile salt hydrolase
genes, properties important for survival and activity in the gastrointestinal tract
(Klaenhammer et al., 2008). The bacterium has shown no adhesion to human intestinal
cells in an in vitro system (Elo et al., 1991; Kleeman et al., 1998). However, regular
yogurt consumption can be a contributing factor to the establishment and survival of L.
bulgaricus in upper and lower gastrointestinal tract (Lick et al., 2001; Mater et al., 2005;
Elli et al., 2006; Vieira et al., 2008). Additionally, a careful setup of the analytic
procedures can improve the reliability of studies regarding the survival of yogurt starters
as has been shown by Elli et al. (2006). The adhesion of some strains with known
probiotic activity like Bifidobacterium lactis Bb12 can be significantly increased in the
presence of L. delbrueckii subsp. bulgaricus when tested in vitro (Ouwehand et al., 2000)
which, in the context of human microbiota, may highlight synergism among healthy
bacteria.
Evidence of plausible probiotic activity of yogurt starter bacteria has been accumulated
predominantly from in vitro studies. The proposed mechanisms of probiotic activity of L.
bulgaricus include: 1) antagonism with pathogens by competition for binding sites and/or
inhibition of intracellular signaling pathways; 2) stimulation of the mucosal immune
system and augmentation of the host defense against pathogenic bacteria and foreign
antigens (Nagafuchi et al., 1999).
L. bulgaricus probiotic activity can be ascribed to its ability to produce substances with
antimicrobial properties. Lactobacilli are known to inhibit the growth of pathogenic
bacteria, possibly by producing inhibitory compounds such as organic acids, hydrogen
peroxide and bacteriocins (Jacobsen et al., 1999; Loessner et al., 2003). Bulgarican,
lactobulgarican, lactobacillin EG4, lactacin A and B are bacteriocins defined in this
species (Reddy et al., 1984; Abdel-Bar et al., 1987; Giraffa et al., 1989; Toba et al., 1991;
Nettles and Barefoot, 1993). Evaluating the cultural conditions with respect to bacteriocin
32
production Balasubramanyam and Varadaraj (1998) have shown that bacteriocin

production is strain dependent and can occur from the logarithmic phase through the early
stationary phase at the optimal growth temperature, 37 - 45C and an acidic pH range
between 4.0 -5.0 (Reinheimer et al., 1990). Bacteriocins are proteinaceous in nature and
stable at 75 C for 30 min. Their inhibition spectrum is narrower than that of antibiotics
(McAuliffe et al., 2001; Morency et al., 2001) and their activity is mainly targeted against
closely related species. A small (3.6-6 kDa) heat stable bacteriocin containing 29 amino
acids from L. bulgaricus has shown inhibitory activity against Listeria monocytogenes,
Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Yersinia enterocolitica
and Y. pseudotuberculosis (Miteva et al., 1998). In light of fermented food industry
bacteriocin producing strains can be effectively used as food biopreservatives. On the
other hand, the deleterious effect of pathogen byproducts on host cells may be diminished
in the presence of L. bulgaricus. A bioactive component released by a L. bulgaricus LDB
B-30892 was capable of inhibiting or deactivating the exotoxins released by C. difficile
thus protecting Caco-2 cells from C. difficile-mediated cytotoxicity (Banerjee et al., 2009).
Pretreatment with L. bulgaricus prior to infection with E. sakazaki, known for its ability to
stimulate the production of NO leading to apoptosis of IEC-6 cells, was effective in
preserving enterocyte integrity both in vitro and in vivo (Hunter et al., 2009). Furthermore,
viable L. bulgaricus cells can prevent TLR4 signaling activation and IL-8 production
mediated by H. pylori in vitro, thus attenuating pathogenic potential of the latter species
(Zhou et al., 2008).
However, so far L. bulgaricus has not been studied with respect to the inhibition of
common oral pathogens.
Immunomodulatory activity has been assessed both in in vitro and in vivo experiments.
The mucosal immune activation is an extremely important characteristic for the selection
of probiotic bacteria (Dogi et al., 2008). Yogurt bacteria may potentiate the production and
the release of IFN- by immunocompetent cells and thereby modulate the host immune
response (DeSimone et al., 1986; Makino et al., 2006). L. bulgaricus strains can induce
cytokine (TNF-a, IL-6, IL-2, and IL-5) secretion in cultured macrophages and T-cells
which play a central role in cell-mediated and humoral immunity (Marin et al., 1998). An
immunostimulatory oligonucleotide sOL-LB17 found in L. delbrueckii supsp. bulgaricus
strain NIAI B6 could substantially bind to B-cells increasing the number of CD69 positive
cells in the Peyer`s patches (Kitazawa et al., 2003).
Consumption of yogurt has been shown to induce measurable health benefits like
strengthening of gut barrier function and prevention of intestinal infections; prevention of
antibiotic-associated diarrhea and immunomodulation (Pereyra and Lemannier, 1993;
Trapp et al., 1993; Meydani and Ha, 2000; Hickson et al., 2007; Zeng et al., 2008).
Positive correlation has been observed in the presence of live bacteria when compared
with products with heat-killed bacteria (Gilliland and Kim, 1984; Savaiano et al., 1984;
Dewit et al., 1988; Lerebours et al., 1989; Van de Water et al., 1999; Rizkalla et al., 2000).
Binding of free bile acids by cells of yogurt starter culture bacteria can even be considered
as a favorable anti-hypocholesterolemic effect of these species (Pigeon et al., 2002).
Moreover, radical scavengers produced in the culture of L. delbrueckii subsp. bulgaricus
2038 may have a preventive effect on the oxidation of LDL (Terahara et al., 2000).
33
According to current scientific concepts, Guarner et al. (2005) have proposed yogurt
starter cultures to be regarded probiotics if a beneficial physiological effect can be
obtained by consumption of the live cultures and the benefit is substantiated appropriately
in human studies.
2.5 Issues of safety in the oral health perspective

The growing market of functional foods and widespread use of probiotics has raised the
question of their possible health risks. Although lactobacilli and bifidobacteria are
ubiquitous in fermented dairy products and possess a GRAS status, there is always the
danger that prolonged probiotic intake may cause bacteraemia or endocarditis, transfer
antibiotic resistance, and to have detrimental metabolic effects in general (Marteau, 2002;
Land et al., 2005; Snydman, 2008, Liong, 2008; Agostoni et al., 2008). However, results
from clinical studies have demonstrated that probiotics are well tolerated by various
patient groups (Millar et al., 1993; Majamaa and Isolauri, 1997; Pedone et al., 1999;
Rosenfeldt et al., 2003; Viljanen et al., 2005) with only a few cases with clinically
manifested side effects (Kirjavainen et al., 2003). Hammerman et al. (2006) have
concluded that the benefits of probiotics outweigh their potential danger, but yet particular
concern must be given to immunocompromised patients and patients with severe
conditions (Salminen et al., 2004; Wada et al., 2009). In a randomized, placebo-controlled,
cross-over study with human immunodeficiency virus patients probiotic use of L.
rhamnosus GG was not found to alleviate gastrointestinal symproms or non-infections
diarrhea, but was not associated with any adverse effects or infections and therefore can be
regarded as safe (Saminen, 2006).
Although probiotics have proven effective against caries pathogens lactobacilli themselves
may associate with caries progression. Some strains of Lactobacillus spp., together with S.
mutans have been implicated in caries development (Montalto et al., 2004). The
production of organic acids from dietary sugars is a leading factor also in dentin caries
progression (Bradshaw and Marsh, 1998). Metabolism and acid production by probiotic
lactobacilli anticipated to exert their properties in the mouth should not favor caries
induction. Adhesion of two probiotics L. casei Shirota and L. acidophilus in an artificial
caries model have shown inconclusive results about the potential of those species in caries
progression; lactobacilli counts were higher in distilled water than in dentin samples under
the terms of the study (Lima et al., 2005). A probiotic L. salivarius LS 1952R
administered to rats in five consecutive days possessed an inherent cariogenic activity
after adherence to tooth surface and enhanced cariogenicity of S. mutans (Matsumoto et
al., 2005). Reproducing oral biofilm model Pham et al. (2009) have observed that L.
salivarius W24 could establish itself in the biofilm if added simultaneously with the
inoculum and it could lower the pH of sucrose-exposed microbiota. These findings
indicate that once established in oral microbiota in the presence of sucrose L. salivarius
W24 might increase the cariogenic potential of the oral microbial community.
Six commercially available lactobacilli, L. plantarum 299v, L. plantarum 931, L.
rhamnosus GG, L. rhamnosus LB12, L. paracasei F19, and L. reuteri were assessed for
34
acid production from various sugars and sugar alcohols (Hedberg et al., 2008). Among
them, L. plantarum strains had the highest activity fermenting glucose, fructose, lactose,
sucrose, maltose, trehalose, and arabinose. Fermentation of glucose, fructose, mannitol,
and trehalose by L. rhamnosus GG resulted in pH values between 5.2 and 6.8 following
24h incubation. L. paracasei and L. plantarum displayed very slow fermentation and pH
values reaching 5.2 6.8 after 72h incubation. The inability of L. rhamnosus strains, L.
paracasei F19 and L. reuteri to ferment sucrose adds valuable information about relative
safety of these probiotic strains in the caries-prophylactic perspective. Another study
addressing sugar fermentation has shown a strain-dependent pH drop and the decrease was
the fastest with glucose for all fourteen strains tested, thus highlighting the acidogenic
potential of probiotics (Haukioja et al., 2008). Bearing in mind the life long tradition of
fermented dairy food consumption without deleterious side effects it can be anticipated
that probiotic administration in a milk product is safer than if given in juice without added
calcium and phosphorus (Meurman, 2009).
Probiotics host tissues cross-talk is another aspect of concern. Epithelial cells play
essential role in providing innate defense against microbial challenge through the
production of antimicrobial molecules, as well as cytokines and chemokines necessary for
leukocyte recruitment (Kagnoff and Eckmann, 1997). Studies in gastrointestinal tract have
shown very low induction of pro-inflammatory cytokines after probiotic challenge (OrtizAndrellucchi et al., 2009; Selvam et al., 2009; Wang et al., 2009). The reaction was
markedly strain-dependent (Medina et al., 2007). A significant reduction of TNF- and IL8 levels in gingival crevicular fluid has been observed after two weeks intake of a chewing
gum with Lactobacillus reuteri ATCC 55730 and ATCC PTA 5289 (Twetman et al.,
2009). Nonetheless, more specific studies are called for the evaluation of safety with the
emergence of new probiotic candidates in the oral cavity.
35
3 Hypotheses and aims of the study

The main objective of this thesis was to assess in vitro the yogurt starter bacterium L.
delbrueckii subsp. bulgaricus for probiotic activity with relevance to the mouth. The final
goal would be to evaluate its suitability for oral cavity applications.
Our working hypothesis was that with the daily intake of yogurt starter L. delbrueckii
subsp. bulgaricus some mechanisms in the development of dental caries and periodontal
diseases are positively affected.
The specific aims of the studies were to assess:
1. the antimicrobial activity of L. delbrueckii subsp. bulgaricus strains against
various
oral
pathogens
(oral
streptococci,
Aggregatibacter
actinomycetemcomitans,
Porphyromonas
gingivalis,
Fusobacterium
nucleatum);
2. the ability of dairy L. delbrueckii subsp. bulgaricus strains to adhere to salivacoated surfaces and to evaluate whether this species might affect the adhesion
of oral streptococci in vitro;
3. the proteolytic activity on human gelatinases of L. delbrueckii subsp.
bulgaricus strains isolated from yogurt, thus addressing the issue of safety
which is a prerequisite for further research on the role of this species on oral
health;
4. the epithelial cell response after stimulation with L. delbrueckii subsp.
bulgaricus and the interference of the species on cytokine response provoked
by P. gingivalis.
36
4 Materials and methods
4.1 Bacterial strains and culture conditions
4.1.1 Lactic acid bacteria

Strains of lactic acid bacteria used in the studies and their culture conditions are listed in
Table 5. L. delbrueckii subsp. bulgaricus strains were kindly provided by LB Lactis
(Scientific-Applied Laboratory for Starter Cultures and Probiotic Products, Plovdiv,
Bulgaria) culture collection in milk medium. They were subcultured in de Man, Rogosa
and Sharpe (MRS) broth at pH 6.4 at 37C in 5% CO2 atmosphere for 24 h. The
lactobacilli were verified by Gram staining and carbohydrate fermentation patterns (API
50 CHL, BioMerieux, Lyon, France). The strains were maintained as frozen stock in
10% skim milk at -70C between different studies.
Table 5. Lactic acid bacteria used in the studies and their culture conditions
Strain
Origin
Growth
Atmosphere
medium
L.
bulgaricus
LBL-12
Laboratory
Incubation
Article
time
MRS
5% CO2
O/N
I, II, III
MRS
5% CO2
O/N
I, II, III
MRS
5% CO2
O/N
I, II, III
MRS
5% CO2
O/N
I, II, III, IV
collection, LB
Lactis,
Bulgaria
L.
bulgaricus
LBL-22
Laboratory
collection, LB
Lactis,
Bulgaria
L.
bulgaricus
LBL-6
Laboratory
collection, LB
Lactis,
Bulgaria
L.
bulgaricus
LBL-83
Laboratory
collection, LB
Lactis,
Bulgaria
37
L.
bulgaricus
LBL-9
Laboratory
MRS
5% CO2
O/N
I, II, III
MRS
5% CO2
O/N
I, II, III, IV
MRS
5% CO2
O/N
I, II, III
MRS
5% CO2
O/N
I, II, IV
MRS
5% CO2
O/N
I, II
MRS
5% CO2
O/N
I, II, IV
MRS
5% CO2
O/N
II, IV
MRS
5% CO2
O/N
II, IV
MRS
5% CO2
O/N
II, IV
MRS
5% CO2
O/N
II
collection, LB
Lactis,
Bulgaria
L.
bulgaricus
LBL-11
Laboratory
collection, LB
Lactis,
Bulgaria
L.
bulgaricus
LBL-23
Laboratory
collection, LB
Lactis,
Bulgaria
L.
bulgaricus
LBL-10
Laboratory
collection, LB
Lactis,
Bulgaria
L.
bulgaricus
LBL-13
Laboratory
collection, LB
Lactis,
Bulgaria
L.
bulgaricus
LBL-42
Laboratory
collection, LB
Lactis,
Bulgaria
L.
bulgaricus
LBL-3
Laboratory
collection, LB
Lactis,
Bulgaria
L.
bulgaricus
LBL-20
Laboratory
collection, LB
Lactis,
Bulgaria
L.
bulgaricus
LBL-39
Laboratory
collection, LB
Lactis,
Bulgaria
L.
bulgaricus
LBL-43
Laboratory
collection, LB
38
Lactis,
Bulgaria
L.
bulgaricus
LBL-81
Laboratory
MRS
5% CO2
O/N
II, IV
MRS
5% CO2
O/N
IV
Ltd.,
MRS
5% CO2
O/N
I, III
Ltd.,
MRS
5% CO2
O/N
I, II, IV
Ltd.,
MRS
5% CO2
O/N
Ltd.,
MRS
5% CO2
O/N
Yakult, Tokyo,
MRS
5% CO2
O/N
collection, LB
Lactis,
Bulgaria
L.
bulgaricus
LBL-80
Laboratory
collection, LB
Lactis,
Bulgaria
L.
bulgaricus
ATCC 11842
Valio
Helsinki,
Finland
rhamnosus
GG
ATCC
Valio
Helsinki,
53103
Finland
L.
Valio
rhamnosus
Lc705
Helsinki,
Finland
L. casei 921
Valio
ATCC 344
Helsinki,
Finland
L. casei Shirota
Japan
Abbreviations: O/N: over-night (16-18h).
4.1.2 Oral bacteria

Oral bacteria used in studies I and II, their origin and growth media are listed in Table 6.
Table 6. Oral bacteria used in the studies.
Strain
Origin
Growth
Atmosphere
medium
S. constellatus ATCC
Incubation
Article
time
ATCC
BHI
5% CO2
24 h
ATCC
BHI
5% CO2
24 h
27823
S. intermedius ATCC
39
27335
S. mitis ATCC 33399
ATCC
BHI
5% CO2
24 h
S. mutans ATCC 25175
ATCC
BHI
5% CO2
24 h
S. oralis ATCC 35037
ATCC
BHI
5% CO2
24 h
S.
ATCC
ATCC
BHI
5% CO2
24 h
ATCC
ATCC
BHI
5% CO2
24 h
ATCC
ATCC
BHI
5% CO2
24 h
ATCC
TS
5% CO2
24 h
ATCC
TS
5% CO2
24 h
ATCC
TS
5% CO2
24 h
ATCC
TS
5% CO2
24 h
ATCC
TS
5% CO2
24 h
A.
Clinical
TS
5% CO2
24 h
actinomycetemcomitans
isolate
TS
5% CO2
24 h
TS
5% CO2
24 h
TS
5% CO2
24 h
Brucella agar
Anaerobic
72 h
sobrinus
33478
S.
salivarius
13419
S.
anginosus
33397
A.
ATCC 29523
A.
ATCC 43718
A.
ATCC 33384
A.
ATCC 37399
A.
ATCC 381
F1000
A.
Clinical
isolate
F 296
A.
Clinical
isolate
F 731
A.
Clinical
isolate
F 982
F. nucleatum ATCC
ATCC
40
25586
P. gingivalis Q 710
Clinical
Brucella agar
Anaerobic
72 h
Brucella agar
Anaerobic
72 h
Brucella agar
Anaerobic
72 h
Brucella agar
Anaerobic
72 h
isolate
P. gingivalis Q 282
Clinical
isolate
P. gingivalis Q 118
Clinical
isolate
P. gingivalis Q 677
Clinical
isolate
4.1.3 Cell cultures

Human mucosal keratinocyte cell line Tuija was used in study IV. This cell line has been
obtained from surgical gingival biopsies and cultured in serum-free low calcium
Keratinocyte Basal Medium (KBM) (Salo et al., 1991) and thereafter underwent
spontaneous immortalization due to transfection with human papilloma virus (Pirisi et al.,
1988). Tuija cells were grown in KGM 2 supplemented with 0.15mM CaCl2, 2 ml BPE15, 0.125 ng ml-1 epidermal growth factor, 5 g ml-1 insulin, 0.33g ml-1 hydrocortisone,
10 g ml-1 transferrin, and 0.39 g ml-1 epinephrine, at 37C and passages between 35-45
were used in the study. For separate experiments, cells were seeded at a concentration of 2
x 10 5 cells ml-1 in 24-well tissue culture plates (Nunc, Roskilde, Denmark), and grown at
37C in 5% CO2 until forming a monolayer with approximately 85% confluence.
4.2 Methods
Methods used in the separate studies are listed in Table 7 and described in detail in the
original articles.
Table 7. Methods used in studies I IV.
Method
Described and used in
Agar-overlay inhibitory assay
Streak-line inhibitory test
Quantitative assessment of adhesion of radiolabeled
II
bacteria to saliva-coated surfaces

41
Western blotting
III
SDS-PAGE
III
ELISA
IV
4.3 Study design
4.3.1 Antimicrobial activity against various oral pathogens (I)

To study the inhibitory activity of lactobacilli against renowned and putative oral
pathogens (strains listed in Table 6) the agar overlay and streak line inhibitory assays were
used dependent on the target bacteria.
Agar overlay method as described by Kakessy and Piguet (1970) was used to
determine the inhibitory activity of lactobacilli against oral streptococci and A.
actinomycetemcomitans. The inhibition zones were measured after incubation for 24 h at
37C in 5% CO2. To measure the inhibitory activity the following formula (Coeuret et al.,
2004) was used:
Where, ID is the diameter of the inhibition halo, CD is the diameter of the colony.
Scores below 0.5 are defined as slight inhibitory activity, between 0.5 and 1.5 as
intermediate, and scores above 1.5 represent strong inhibition.
Streak-line inhibitory activity test was performed according to Annuk et al. (2003) to
study the inhibitory activity of lactobacilli against P. gingivalis and F. nucleatum strains.
After 72 h incubation the width of the zone of inhibition (mm) extending from the target
bacteria to the lactobacilli streak line was measured (Mikelsaar et al., 1987).
4.3.2 Adhesion to saliva-coated surfaces in vitro (II)

For adhesion studies the bacteria (listed in Table 5) were radiolabeled by growing the cells
in appropriate broth supplemented with 10 l/ml of [methyl-1,2-3H]thymidine, 122
Ci/mmol (GE Healthcare, Chalfont St Giles, Buckinghamshire, UK) as previously
described (Fernandez et al., 2003).
Unstimulated whole saliva was collected from five healthy individuals who were
instructed not to eat, drink, smoke, or use chewing gum for an hour before the saliva
collection. Informed consent was obtained before the collection began. The saliva was
collected into chilled tubes on ice and clarified by centrifugation (14,000 g for 20 min at
4C). The pooled samples were divided into aliquots and frozen at -20C before the
adhesion assays.
42
4.3.2.1 Adhesion to sHA beads

Spheroid HA beads (Macro-Prep Ceramic Hydroxyapatite TYPE II 80 m, Bio-Rad
Laboratories, Hercules, CA) were equilibrated for 2 h in buffered KCl (0.05 m KCl
containing 1 mm KH2PO4, 1mm CaCl2 and 0.1 mm MgCl2 at pH 6.5). 100 l saliva was
added per well and the mixture was incubated for 1 h at 37C. After three washings with
buffered KCl (200 l/well) 100 l radioactive bacterial suspension was added to each well
and incubated with shaking (50 r.p.m.) for 1 h. The radioactivity was measured by liquid
scintillation counter (Winspectral 1414, Wallac, Turku, Finland). The adhesion ratio (%)
of bacteria was calculated by comparing the radioactivity of the adhered bacteria to the
radioactivity of the added bacteria.
4.3.2.2 Adhesion to saliva-coated microtiter plates

Adhesion to human saliva was assessed according to the method studying adhesion to
intestinal mucus as described earlier by Ouwehand et al., (2001). Saliva was immobilized
passively overnight at 4C in 96-well polystyrene microtiter plates (Maxisorp, Nunc,
Roskilde, Denmark; 100 l/well). Bacterial suspensions were added (100 l/well) and
bacteria were allowed to adhere at 37C for 1 h. Lactobacilli pretreated with lysozyme
(0.05 mg/ml in phosphate-buffered saline (PBS), pH 6.2) were assessed for their ability to
adhere to saliva.
4.3.2.3 Adhesion to solvents

Microbial adhesion to n-hexadecane was measured according to the method of Rosenberg
et al. (1980). A detailed description of the method is given in study III.
4.3.2.4 Effect of lactobacilli pretreatment on streptococcal adhesion in vitro

To study the effect on adhesion of S. sanguinis ATCC 10556 after Lactobacillus
pretreatment of saliva-coated MaxiSorp plates, non-radiolabeled L. delbrueckii subsp.
bulgaricus strains were allowed to adhere to immobilized saliva for 1 h at 37C. After two
washes with HEPESHanks buffer, 100 l streptococcal suspension was added per well
and incubated for 1 h at 37C and the adhesion experiment was performed as already
described.
43
4.3.3 Proteolytic activity on human gelatinases (III)

The proteolytic activity of different L. bulgaricus strains (listed in Table 5) on human
progelatinase B (pro-MMP-9) was evaluated based on a protocol used for assessing the
gelatinolytic activity of defined oral pathogens (Grayson et al., 2003).
Lactobacilli were grown in de Man, Rogosa and Sharpe broth at pH 6.4 (MRS broth,
LAB MTM, IDG Ltd., Lankashire, UK) at 37C in 5% CO2 atmosphere. Cells were
harvested by centrifugation at 5 000 g for 20 min, and the supernatants were dialyzed
against distilled water for 2 h at 4C. Harvested cells were washed twice with PBS, pH
7.4, suspended in 1 mL of PBS. Prior to use they were sonicated on ice to disrupt the cells.
Both cell fractions and the supernatant fractions were used in this study.
The presence of gelatinolytic proteases was assayed with the use of an enzymography
in 0.75-mm-thick 11% SDS-PAGE gels impregnated with 1 mg mL-1 gelatin, as described
in Study III. White zones of lysis indicating gelatine degradation were revealed by
staining with 1% Coomassie Brilliant Blue.
The molecular forms of MMP-9 were detected by a modified (Sorsa et al., 1997) ECL
Western blotting kit according to protocol recommended by the manufacturer (GE
Healthcare, Amersham, UK).
To determine the inhibitory effect of different synthetic MMP inhibitors on L.
bulgaricus proteases, Ilomastat (Chemicon International Inc., CA, USA), EDTA (Merck,
KGaA, Dramstadt, Germany), CMT3, CMT308 (Collagenex Inc., Newton, PA, USA),
CTT1 (Koivunen et al., 1997) and a serine protease inhibitor, Pefabloc (Boehringer
Mannheim GmbH, Manheim, Germany), were employed in this study. The MDPFzymography was assayed as previously to detect the residual gelatinolytic activity.
4.3.4 Epithelial cell response to L. delbrueckii subsp. bulgaricus
4.3.4.1 Induction of IL-8 and TNF- secretion

Lactobacilli in KGM 2 or culture supernatants (1 ml) were added to the epithelial cell
monolayers and incubated for 6h and 24h at 37C in 5% CO2. KGM 2 alone served as a
negative control. At each time point 500l of the cell culture medium was removed and
centrifuged to obtain debris-free supernatant. Collected supernatants were stored at -20C
until ELISA assessment.
4.3.4.2 Epithelial cell response to P. gingivalis after lactobacilli pretreatment

After 24h incubation with lactobacilli the epithelial cells were washed twice with PBS and
P. gingivalis in KGM 2 was added to a volume of 1 ml to each well. The epithelial cell
monolayers were then cultured at 37C in 5% CO 2. After 1, 2 and 24h 500l supernatants
were collected for ELISA analyses.
44
4.4 Statistical analyses

All experiments were run at least in duplicate and scores are presented as means SEM.
Differences were considered significant when P<0.05. Appropriate parametric and nonparametric tests employed are described in detail in studies I-IV.
45
5 Results and discussion

The present series of studies addressed several key aspects of probiotic activity to be
assessed with respect to possible oral cavity applications.
5.1 Inhibition of oral pathogens (Study I)

Thirty lactobacilli strains, 11 of which were L. bulgaricus, were assessed for their
inhibitory activity against 23 strains of oral pathogens.
A. actinomycetemcomitans strains were the most susceptible to the inhibitory activity
of the lactobacilli tested. The inhibition varied significantly from slight to strong (p<0.05).
Four L. bulgaricus strains, namely L. bulgaricus LBL-9, L. bulgaricus LBL-11, L.
bulgaricus LBL-23, and L. bulgaricus LBL-83, showed the most pronounced inhibitory
activity among all strains tested (EIR>2). No difference was observed in growth inhibition
between the clinical isolates of A. actinomycetemcomitans vs. commercial strains. Table 8
presents the inhibitory activity of L. bulgaricus strains against A. actinomycetemcomitans.
Our results are in agreement with the results by Koll-Klais et al., (2005) who reported that
homo-fermentative lactobacilli expressed significant antimicrobial activity against
periodontal pathogens. L. bulgaricus is an obligate homofermentative and most of the
strains strongly inhibited A. actinomycetemcomitans.
Table 8. Inhibition of clinical and commercial isolates of A. actinomycetemcomitans by L.
delbrueckii subsp. bulgaricus strains tested.
Strain
Aggregatibacter actinomycetemcomitans
ATTC
ATCC
ATCC
ATCC
ATCC
Mean
29523
43718
33384
37399
381
1000
296
731
982
SD
2.4
1.4
1.2
1.9
1.3
1.3
1.8
1.8
1.5
1.60.3
2.2
2.8
3.2
2.4
1.4
1.5
1.5
2.1
2.1 0.5
2.9
0.8
0.8
1.2
1.1
0.9
1.4
1.1
1.3
1.6 1.6
0.5
1.3
0.1
0.6
0.3 0.4
L.
bulgaricus
365
L.
bulgaricus
LBL-23
L.
bulgaricus
LBL-12
L.
46
bulgaricus
LBL-22
L.
3.2
0.3
0.6
1.1
bulgaricus
0.60.0
5
LBL-6
L.
2.2
2.3
2.8
2.5
0.9
2.5
1.1
1.9
2.9
2.10.7
0.5
2.3
1.7
1.3
1.2
0.7
1.30.9
4.5
3.8
2.1
1.3
1.1
1.1
2.5
2.2
2.51.3
1.6
4.2
2.2
2.1
1.7
1.6
2.1
1.2
2 0.8
2.2
3.8
3.5
3.3
1.3
2.4
1.3
2.2
2.40.9
3.5
2.8
3.7
4.4
1.5
1.6
1.8
1.8
1.6
2.51.1
bulgaricus
LBL-10
L.
bulgaricus
LBL-13
L.
bulgaricus
LBL-83
L.
bulgaricus
LBL-42
L.
bulgaricus
LBL-9
L.
bulgaricus
LBL-11
Oral streptococci showed various patterns of susceptibility to lactobacilli. In general the

average inhibitory activity of lactobacilli against streptococcal species was low (EIR =
0.5). Among the lactobacilli tested only strains of L. bulgaricus inhibited the growth of
oral streptococci. The pattern of inhibitory activity of L. bulgaricus strains against
streptococcal species is given in Figure 2. In the present study S. mutans was strongly
inhibited by single L. bulgaricus strains (L. bulgaricus LBL-23 being the strongest).
Strahinic et al. (2007) and Koll-Klais et al. (2005) have shown that S. mutans is
susceptible to growth inhibition by various oral lactobacilli. Considering the common use
of yogurt in the diet of many populations dairy strains may indeed affect the composition
of oral biofilm formation already in early childhood if the dairy starter bacteria interact
with commensal microflora, thus altering its cariogenic potential.
47
S. oralis
S.mutans
S. sobrinus
S. mitis
S. constellatus
S. intetrmedius
S. salivarius
S. anginos
1.8
1.6
Effective inhibitory ratio
1.4
1.2
1
0.8
0.6
0.4
0.2
0
LBL-23
LBL-22
LBL-6
LBL-10
LBL-13
LBL-83
LBL-42
LBL-9
LBL-11
L. bulgaricus strains
Figure 2. Effective inhibition ratio against oral streptococci by L. bulgaricus.
The mechanisms by which L. bulgaricus strains inhibited oral pathogen growth are not
fully understood. Lactobacilli may exert their antibacterial activity through the production
of organic acids (lactic and acetic acid) and other metabolites such as hydrogen peroxide
and diacetyl, or specific bactericidal or bacteriostatic peptides and proteins (De Vuyst et
al., 1994). We observed that when lactobacilli were grown on MRS agar with normal
glucose content, instead of 0.2% glucose agar, growth inhibition of both the streptococci
and A. actinomycetemcomitans was more pronounced. Koll-Klais et al. (2005) reported the
same which indicates that the availability of substrate for fermentation seems to be one of
the essential factors for the antimicrobial activity. We have found that culture supernatants
of lactobacilli possessed no antimicrobial activity against streptococci and A.
actinomycetemcomitans when using the well-diffusion or paper-disk assays according to
Drago et al., (1997). Hence, the inhibitory mechanisms may be cell bound functions.
Streak line inhibition test used to study the effect of lactobacilli against P. gingivalis
and F. nucleatum showed low susceptibility of these bacteria. There was no inhibitory
activity observed among L. bulgaricus strains against either P. gingivalis or F. nucleatum.
The inhibitory pattern varied distinctly between the lactobacilli tested. However, there
was no single Lactobacillus strain to demonstrate growth inhibition against all four oral
pathogen species used. Thus, no single Lactobacillus species can be used in combating
oral pathogens in broader sense and several species need to be considered when selecting
oral probiotics.
48
5.2 L. bulgaricus adhesion to saliva-coated surfaces (Study II)

Adhesion of bacteria to host surfaces is regarded of major importance in contributing to
permanent, or even transient, establishment of probiotic species in any environmental
niche. In the present study we focused on the bacterial adhesion to human saliva that is the
main fluid overlying oral surfaces. Presumably probiotic bacteria that express good
binding ability to salivary pellicle may also be able to colonize the oral cavity.
Saliva-coated HA beads have been commonly used as an in vitro model to
quantitatively study adhesion of radiolabeled bacteria because the surface properties are
similar to those of tooth enamel (Gibbons et al., 1982). Adhesion to sHA varied between 1
to 17%, and L. bulgaricus LBL-39 exhibiting values comparable to that of the reference
strains S. sangunis. S. sanguinis is the first colonizer on tooth surfaces in vivo and its
ability to adhere to sHA make it a suitable model for dental adhesion studies.
Generally, the adhesion of most L. delbrueckii subsp. bulgaricus strains to sHA was
low (<5%) under the present experimental conditions. The adhesion to saliva-coated
Maxisorp plates ranged between 3 and 22%, with LBL-39 exhibiting the strongest ability
to adhere.
A significant increase in the adhesive properties was observed when the strains were
pretreated with lysozyme (P < 0.05); results are shown in Figure 3.
Figure 3. Adhesion of L. bulgaricus to saliva-coated Maxisorp plates after lysozyme

pretreatment.
Tellefson and Germaine (1986) have found that lysozyme promoted the adherence of
some oral streptococci (S. sanguinis) to sHA. The role of lysozyme pretreatment on
probiotic properties has recently been also addressed as a factor improving the
immunostimulatory effect of probiotic species (Bu et al., 2006).
Cell surface hydrophobicity has been considered a valuable reference when evaluating
the adhesive properties of microorganisms. High hydrophobicity correlated with marked
adhesion (Wadstrm et al., 1987). The assessment of cell surface hydrophobicity might be
used as a test for studying adhesive properties of bacteria because this characteristic has
been reported to objectively reflect microbial adhesion (Ellepola et al., 2001; Wadstrm et
49
al., 1987). By measuring adhesion to n-hexadecane we observed that the strains

investigated showed various patterns of interaction with the organic solvent, as shown in
Figure 4. The Lactobacillus strain LBL-39 which had shown the most pronounced
adhesive properties to saliva-coated surfaces again displayed the strongest adhesive
potential.
Figure 4. Adhesion of L. bulgaricus to n-hexadecane.
As S. sanguinis and lactobacilli were able to adhere to saliva-coated surfaces we

hypothesized that these two species may compete when present together. However, the
adhesion of S. sanguinis ATCC 10556 was not significantly affected by the pretreatment
of the wells with any of the Lactobacillus strains, as shown in Figure 5.
Figure 5. Adhesion of S. sanguinis ATCC 10556 to saliva-coated microtiter plates

after pretreatment with the lactobacilli studied.
The competitive inhibition for bacterial adhesion sites has been considered as a favorable
mechanism for probiotic action (Fernandez et al., 2003). Despite the fact that lactobacilli
50
adhered to various extents to the immobilized saliva they were not able to affect the
adhesion of the target microorganism tested. It could therefore be concluded that the
salivary receptors are different for dairy strains and S. sanguinis and that pretreatment with
lactobacilli does not block streptococcal adhesion by steric hindrance. Similar results were
observed for other probiotic species that also lacked the capacity to change the adhesive
potential of several skin pathogens (Ouwehand et al., 2003).
Issues of safety demand substantial consideration and in vitro tests are critical when
assessing the mechanisms of probiotic effect with no hazards being imposed on the host
by the use of living microorganisms in therapy. Safety issues of lactobacilli have been
studied by evaluating adhesion to main constituents of extracellular matrix: collagen type
IV and fibrinogen; binding to intestinal mucus; induction of respiratory burst in peripheral
blood monocytes and resistance to serum-mediated killing (Vesterlund et al., 2007). There
were no studies addressing the issues of safety related to screening of putative probiotic
species with the scope of application in the oral cavity. A favorable metabolic activity and
harmless host-bacteria interactions that pose no risk to oral health of the individual must
be considered when putative probiotics are administered in the mouth. L. delbrueckii
subsp. bulgaricus that is essential in yogurt production exerts high hydrolysing activities
towards substrates containing proline, alanylprolylpnitroanilide and prolylp
nitroanilide (Sasaki et al., 1995). It is known that L. delbrueckii subsp. bulgaricus
possesses a complex proteolytic system essential for rapid growth in protein-rich media
(Atlan et al., 1994) and the hydrolysis of milk caseins by means of a cell-wall proteinases
has been extensively studied (Smid et al., 1991; Laloi et al., 1991). A cell-envelopeassociated aminopeptidase characterized as metallo-enzyme with a broad specificity has
been purified from the cell wall of L. bulgaricus, L. lactis, and L. helveticus (Atlan et al.,
1989; Blanc et al., 1993).
5.3 Proteolytic activity on human progelatinase B (proMMP-9)

(Study III)
In the present study we applied a method evaluating the effect of probiotic candidates on
the activation of matrixmetalloproteinases (MMPs), the enzymes responsible for
extracellular matrix degradation and remodeling. Elevated levels of salivary MMPs have
been associated with metabolic activity of various oral pathogens (Ding et al., 1997;
Mntyl et al., 2003; Sder et al., 2006). Thus, the capacity of some microbial species to
convert extracellular matrix enzymes into their active forms might be considered an
inherent virulence factor.
5.3.1 Gelatinolytic activity

Gelatin zymography with labeled substrate enables the detection of type I and type IV
collagenolytic activity. The gelatinolytic activity of all strains tested was very low
compared with positive human saliva controls. Degradation of gelatin was not detected
51
after an 18 h incubation period. However, the prolonged 7-day incubation time yielded
molecular weight bands at the area of 106 kDa and around 150 kDa (Figure 6). There was
no significant difference in the gelatinolytic activity when the different pH values of the
buffers were used. Supernatant samples, although showing only weak proteolytic activity,
were more potent in degrading gelatin than the cell fraction samples which yielded no
visible bands on the UV light picture. No difference was observed among the strains in the
degradation of gelatin.
Figure 6. Gelatinolytic activity of L. delbrueckii subsp. bulgaricus supernatants. All

strains show similar bands of activity after a 7-day incubation period.
Considering the attachment to oral mucosa, it is essential that the microorganism is not
harmful to mucosal cells and extracellular matrix and basement membrane components. A
damaged or disintegrated oral epithelium facilitates a microbial invasion, providing
appropriate environment for further bacterial growth. Most bacterial proteinases, however,
have weak degrading activity against collagen (Okamoto et al., 2004). Once activated
human collagenolytic MMPs might provide suitable substrate for further activity of human
gelatinases or other bacterial proteinases. The test strains investigated in our study
demonstrated very low gelatinolytic activity even after the longest incubation period,
which validates their relative safety as probiotic candidates.
5.3.2 Activation of proMMP-9

ProMMP-9 was incubated for three different time periods with supernatants and cell
fraction suspensions of the L. bulgaricus strains. The conversion of proMMP-9 into its
52
active form was not detected after 24h of incubation as shown by Western blotting with
the anti-MMP-9 antibodies (Figure 7A and 7B).
Figure 7. L. delbrueckii subsp. bulgaricus strains are ineffective in converting

proMMP-9 to its active form as shown on Western blotting images. A. Supernatant fractions; B.
Cell fractions.
MMPs are expressed at low levels in the absence of inflammation, wound healing or other
pathological processes (Woessner, 1991).
MMP-9 and other endogenous proteinases hydrolyze and degrade the fragments of
denatured collagens, for example gelatin, into smaller fragments. It has been shown in
many studies that MMP-9 is a specific marker for periodontal destruction (Ejeil et al.,
2003) and elevated levels of this enzyme are related to the severity of periodontal
breakdown. Referred to as type IV collagenase MMP-9 is particularly implicated in the
degradation of the basement membrane (Reynolds and Meikle, 1997). The proteolysis of
the ECM seems to play an important role in initiating the progression of the inflammatory
process, and thus conversion of proMMPs into their active forms is a crucial step here,
facilitating bacterial adhesion and infection. Studies on the activation of human MMPs
have shown that some bacterial species with clear pathogenic potential are capable of
activation of MMPs. For example, Vibrio proteinase and Pseudomonas elastase have
shown stronger activation of pro-MMP-9 than did APMA (Okamoto et al., 1997).
Furthermore, pro-MMPs can be activated by a variety of mechanisms that include
proteinases such as plasmin; thiol-oxidizing agents, e.g., HgCl2 and N-ethylmaleimide;
53
low pH; and heat treatment (Vise and Nagase, 2003). MMPs are secreted as proenzymes
and their activity is low in intact normal tissues but could undergo activation by a broad
range of stimuli (Sorsa et al., 1997; Johnson et al., 1998; Potempa et al., 2000; Okamoto et
al., 2004). A key event in the activation of proMMPs is the removal of the propeptide
domain in their structure that usually consists of ca. 80 amino acid residues (Nagase et al.,
1990). Lactobacillus bulgaricus strains in our study were incapable in converting the
proMMP-9 to the 6080 kDa forms considered active and did not show any activity at the
region of the molecular mass consistent with protease IV.
5.3.2 Proteolytic activity of L. bulgaricus strains in the presence of

synthetic MMP inhibitors
To investigate if the synthetic inhibitors of MMPs affect the gelatinolytic activity of
bacterial proteases, the L. bulgaricus strains tested were incubated with five different
synthetic MMP inhibitors and Pefabloc. No significant changes in gelatinolytic activity
were observed on Coomassie Brilliant Bule stained gels. Synthetic MMP inhibitors and
Pefabloc did not affect the proteolytic activity of the supernatants or the cell fraction
suspensions of the L. bulgaricus strains investigated.
The administration of synthetic inhibitors of MMPs is considered a therapeutic
approach in the treatment of different pathological conditions in which elevated levels of
MMPs are regarded as key factors in inflammation and tissue breakdown. The preserved
and unaffected proteolytic activity of the test strains after addition of different synthetic
MMP inhibitors and Pefabloc in the test system might additionally benefit the anticipated
probiotic effect of those microorganisms. Consequently, a simultaneous administration of
potential probiotics and inhibitors of MMPs should not be regarded contradictory when
potential new treatment modes for infectious diseases are being considered.
5.4 Epithelial cell lactobacilli interactions (Study IV)

In vitro experiments as conducted here are the first step in the evaluation of safety aspects
when the oral cavity is exposed to high numbers of lactobacilli. The integrity of the
epithelial lining of the oral cavity is part of the innate defense and serves as an effective
barrier against various microorganisms.
The ability of oral epithelial cells to secrete proinflammatory cytokines, IL-8 and TNF, in response to five different lactobacilli species at two different concentrations, 106
CFU ml-1 and 109 CFU ml-1, was examined. The viability of cultured epithelial cells
remained above 85% during the whole set of experiments.
In the present study the strongest induction of IL-8 secretion was observed with live
bacterial samples at the higher concentration (109 CFU ml-1) compared to heat killed
bacteria (p < 0.05). The increased levels of IL-8 were concentration dependent. Heat killed
bacterial samples at concentration of 106 CFU ml-1 were stronger inducers of IL-8 than
heat killed bacteria at 109 CFU ml-1 (p < 0.05). One strain, namely L. bulgaricus LB-86,
54
induced significantly lower secretion of IL-8 compared with A. actinomycetemcomitans

positive controls (p < 0.05). The remaining strains within this group showed IL-8 values
similar to those measured for A. actinomycetemcomitans at the 6h incubation-point. Heat
killed L. bulgaricus LB-39, L. bulgaricus LB-3, L. bulgaricus LB-11, L. bulgaricus LB42, L. bulgaricus LB-86 induced significantly higher levels of IL-8 compared to their live
counterparts 6h after co-culturing with the epithelial cells. Figure 8 shows the dynamics
of IL-8 secretion within 24h of co-culturing of lactobacilli with the epithelial cells.
Bacterial culture supernatants of all the strains tested led to undetectable levels of IL-8 in
the culture medium. Furthermore, the addition of P. gingivalis to the epithelial cells
pretreated with lactobacilli showed an almost immediate disappearance of any detectable
levels of IL-8 in culture medium.
Figure 8. Levels of IL-8 secreted after co-culturing of oral epithelial cells with heat
killed and live lactobacilli strains at two different concentrations ( OD = 0.1 and OD = 0.5,
corresponding to 106 and 109 CFU ml-1). Supernatants were collected at 6 and 24h.
After co-culturing with lactobacilli the epithelial cells responded with different
concentrations of TNF- secreted in the culture medium. Generally, the concentration of
TNF- was low in most cases. Bacterial culture supernatants were unable to stimulate
cytokine secretion. The higher bacterial concentrations (109 CFU ml-1) led to a significant
difference between the live and heat killed bacteria (p <0.05). L. bulgaricus LB-39
induced a significant increase of TNF- , whereas seven L. bulgaricus strains produced no
effect. The addition of P. gingivalis led to a significant increase in TNF- in the culture
55
medium and the concentration increased during the first 2 hours of incubation, whereas at
the end of the experiment the detected values were lower. When the epithelial cells were
pretreated with lactobacilli prior to the P. gingivalis addition the concentration of secreted
TNF- was lower than when P. gingivalis was added alone to the cells maintained in the
culture medium.
Hence in our present study we analyzed the secretion of two common proinflammatory
cytokines that are generally associated with inflammation. Cytokines are secreted proteins
that are responsible for many of the cellular responses of the innate and adaptive
immunity, and thus function as the "messenger molecules" of the immune system. IL-8
and TNF- are released by the oral epithelium in response to fungal or bacterial infection
and they trigger further cellular responses. Cytokine expression induced by lactobacilli at
various mucosal sites has been investigated in animals, human biopsy specimens, as well
as in monolayer cell culture models. The inhibition of secretion of IL-4, IL-5, and IL-8 has
been defined as a property of many strains of lactobacilli (Carbo et al., 2002; Pochard et
al., 2002). However, the oral epithelium has not yet been investigated regarding cytokine
expression after probiotic challenge. Because of the emerging concern of probiotic safety,
especially in cases with immunocompromised patients, the results obtained by us merit
particular interest. It is noteworthy to point out that probiotic properties do not always
require administration of live bacteria. Additionally, fermented dairy products are
common vehicles for probiotics and the oral cavity obviously serves as the first site where
these bacteria can exert their effects. Subsequently our results provide further evidence
showing that higher doses of live probiotic or putative probiotic species may induce IL-8
secretion similar to what was observed with the periodontal pathogen A.
actinomycetemcomitans.
By including P. gingivalis in the study we investigated whether probiotic interaction
with the epithelial cells affects further cell response caused by the P. gingivalis. This
organism is an established periodontal pathogen that in addition to its large array of
virulence factors inducing periodontal tissue damage may also possess a variety of evasion
mechanisms towards host defense. Among others these may lead to altered
polymorphonuclear cell (PMN) function and impaired immune response in general. We
have demonstrated here that the epithelial cells pretreated with lactobacilli produced
pronounced levels of IL-8, shortly after the addition of P. gingivalis suspensions and also
displayed absence of IL-8 in the cell culture supernatant. This phenomenon could be
attributed to the high proteolytic activity of P. gingivalis which causes degradation of
cytokines and chemokines (Calkins et al., 1998; Banbula et al., 1999; Bodet et al., 2005).
A strain dependent pattern on TNF- secretion after P. gingivalis challenge was also
observed. The strongest inducer of TNF- among the L. bulgaricus strains, LB-39, led to
significantly lower levels of TNF- after incubation with P. gingivalis. Attenuated
expression, but not absence of TNF- after the P. gingivalis infection, was observed with
another L. bulgaricus strain, namely LB-80. On the other hand, pretreatment of the
epithelial cells with heat killed L. bulgaricus LB-42 led to six fold increase in TNFconcentrations after P. gingivalis challenge. These results provide evidence to the complex
mechanisms of interaction between lactobacilli and epithelial cells and warrant further
investigations.
56
6 Key findings and conclusions

These series of studies addressed some key characteristics for the evaluation of probiotic
properties of L. delbrueckii subsp. bulgaricus with respect to the oral cavity. The main
findings can be summarised as follows:
1.
2.
3.
4.
Among L. delbrueckii subsp. bulgaricus species there are strains capable of

inhibiting growth of some key oral pathogens, S. mutans and A.
actinomycetemcomitans being the most susceptible to the inhibitory effect.
The adhesive properties of yogurt starter L. delbrueckii subsp. bulgaricus to
saliva-coated surface are comparatively low although single strains
demonstrated adhesive potential similar to that of strongly adhering reference
species.
L. delbrueckii subsp. bulgaricus strains are harmless to main components of
extracellular matrix, being unable to convert proMMP-9 to its active form, thus
highlighting their safety on regulatory enzymes and structures of the host
extracellular matrix.
L. delbrueckii subsp. bulgaricus can induce IL-8 and TNF- after stimulation of
oral epithelial cells in vitro which is strain and concentration dependent. The
addition of P. gingivalis to epithelial cells pretreated with lactobacilli led to
pronounced reduction of cytokine levels in cell culture supernatants probably
due to its high proteolytic activity.
Based on the results of this research it is suggested that among the L. delbrueckii subsp.
bulgaricus species there are strains that could be further studied as probiotics with
eventual health promoting effects in the oral cavity. Furthermore, a combination of several
strains with favorable properties merits further investigation in the oral health perspective.
However, more research is needed to optimize the selection of proper strain/s to be used as
oral probiotics and to decide the best and appropriate means for probiotic administration
into the mouth. Phase I, II, III, and IV clinical trials need then to be conducted.
57
Acknowledgements
These studies were performed at Biomedicum, Institute of Dentistry, University of

Helsinki during the years 2006-2008. I thank the dean, Professor Jarkko Hietanen, for
providing the facilities at my disposal. I received a lot of expert advice while doing the
series of studies and everyone I asked gave generously of their time and knowledge.
I wish to thank all the people who have contributed to the study:
My supervisor, Professor Jukka H. Meurman, is cordially thanked for inviting me to
become part of his research team and introducing me to the enticing world of probiotics.
His consistent supervision, detailed assessment of every step of my work and numerous
discussions accompanying my studies ensured this thesis to be completed. I would like to
express my deep sense of gratitude for the priceless lessons in science I learned working
with him.
I wish to thank Professor Maria Baltadjieva who ignited my belief in the probiotic
activity of yogurt lactobacilli and provided all L. bulgaricus strains studied in my work.
Professor Stoyan Vladimirov is thanked for his support and understanding of my present
work.
Professors Riitta Korpela and Jorma Tenovuo, the official reviewers of the thesis, are
gratefully acknowledged for constructive criticism and insightful comments on the
manuscript.
I am greatly indebted to Kirsti Kari, MSc, Head of the Scientific Laboratory, for her
invaluable instructions and devoted assistance. Her amazing managerial and
communicative skills made me feel at home in the laboratory and fight back homesickness
during my stay in Finland.
Professor Timo Sorsa and colleagues in the dental laboratory illuminated various
problems and issues in both departmental seminars and less formal settings. Many thanks
to you all. My special thanks to docent Taina Tervahartiala, being untiringly helpful from
the beginning of my laboratory work and providing insightful advice on methodological
issues.
I wish to thank Pipsa Kaipainen from Institute of Biomedicine who was very kind to
assist me with cell culture experiments.
I would also like to thank Ritva Keva and Jukka Inkeri, BSc, for their excellent
technical support in the laboratory.
My stay in Biomedicum provided me the opportunity to make unforgettable
acquaintances with colleagues from Finland and abroad and broaden my awareness of
both professional and cultural issues. I am grateful for the relaxing moments during spare
time and the treasured memories I shall never forget.
58
My heartfelt thanks are due to my mother Dora Yancheva for her endless love, support
and encouragement to follow my dreams.
Finally, my deepest thanks go to my husband, Dimitar, for sharing his life with me and
trying to understand and support me during these hardest years of my life. In addition, I
am grateful to our adorable twin-daughters, Lora and Mia, who were my constant source
of faith and inspiration.
Helsinki, March 2009
Iva Stamatova
59
References
Aas JA, Paster BJ, Stokes LN, Olsen I, Dewhirst FE. Defining the normal bacterial flora of the
oral cavity. J Clin Microbiol 2005;43:5721-5732.
Aas JA, Griffen AL, Dardis SR, Lee AM, Olsen I, Dewhirst FE, Leys EJ, Paster BJ. Bacteria of
dental caries in primary and permanent teeth in children and young adults. J Clin Microbiol 2008;
46:1407-1417.
Abdel-Bar N, Harris ND, Rill RL. Purification and properties of an antimicrobial substance
produced by Lactobacillus bulgaricus. J Food Sci 1987;52:411415.
Afolabi OC, Ogunsola FT, Coker AO. Susceptibility of cariogenic Streptococcus mutans to
extracts of Garcinia kola, Hibiscus sabdariffa, and Solanum americanum. West Afr J Med
2008;27:230-233.
Agostini C, Axelsson I, Braegger C, Goulet O, Koletzko B, Michaelsen KF, Rigo J, Shamir R,
Szajewska H, Truck D, Weaver LT. Probiotic bacteria in dietetic products for infants: a commentary
by the ESPGHAN Committee on Nutrition. J Pediart Gastroenterol Nutr 2004;38:365-374.
Ahola AJ, Yli-Knuuttila H, Suomalainen T, Poussa T, Ahlstrm A, Meurman JH, Korpela R.
Short-term consumption of probiotic-containing cheese and its effect on dental caries risk factors. Arch
Oral Biol 2002;47:799-804.
Ahrne S, Nobaek S, Jeppsson B, Adlerberth I, Wold AE, Molin G. The normal Lactobacillus
flora of healthy and rectal oral mucosa. J Appl Microbiol 1998;85:88-94.
Akers HF. Collaboration, vision and reality: water fluoridation in New Zealand (1952-1968). N
Z Dent J 2008;104:127-133.
Aldridge PD, Gray MA, Hirst BH, Khan CM. Who's talking to whom? Epithelial-bacterial
pathogen interactions. Mol Microbiol 2005;55: 655-663.
Altman H, Steinberg D, Porat Y, Mor A, Fridman D, Friedman M, Bachrach G. In vitro
assessment of antimicrobial peptides as potential agents against several oral bacteria. J Antimicrob
Chemother 2006;58:198-201.
Amerongen AV, Veerman EC. Saliva - the defender of the oral cavity. Oral Dis 2002;8:12-22.
Andrews JH, Harris RF. The ecology and biogeography of microorganisms on plant surface.
Annu Rev Phytopathol 2000;38:145180.
Annuk H, Shchepetova J, Kullisaar T, Songisepp E, Zilmer M, Mikelsaar M. Characterization
of intestinal lactobacilli as putative probiotic candidates. J Appl Microbiol 2003;94:403-412.
Anukam KC, Osazuwa E, Osemene GI, Ehigiagbe F, Bruce AW, Reid G. Clinical study
comparing probiotic Lactobacillus GR-1 and RC-14 with metronidazole vaginal gel to treat
symptomatic bacterial vaginosis. Microbes Infect 2006;8:2772-2776.
Ataie-Jafari A, Larijani B, Alavi Majd H, Tahbaz F. Cholesterol-lowering effect of probiotic
yogurt in comparison with ordinary yogurt in mildly to moderately hypercholesterolemic subjects. Ann
Nutr Metab 2009;54:22-27.
60
Atlan D, Gilbert C, Blanc B, Portalier R. Cloning, sequencing and characterization of the pepIP
gene encoding a proline iminopeptidase from Lactobacillus delbrueckii subsp. bulgaricus CNRZ 397.
Microbiology 1994; 140:527-535.
Atlan D, Laloi P, Portalier R. Isolation and characterization of aminopeptidase-deficient
Lactobacillus bulgaricus mutants. Appl Environ Microbiol 1989;55:1717-1723.
Balasubramanyam BV, Varadaraj MC. Cultural conditions for the production of bacteriocin by
a native isolate of Lactobacillus delbruecki ssp. bulgaricus CFR 2028 in milk medium. J Appl
Microbiol 1998;84:97-102.
Bamford CV, d'Mello A, Nobbs AH, Dutton LC, Vickerman MM, Jenkinson HF. Streptococcus
gordonii modulates Candida albicans biofilm formation through intergeneric communication. Infect
Immun 2009;77:3696-3704.
Banbula A, Bugno M, Kuster A, Heinrich PC, Travis J, Potempa J. Rapid and efficient
inactivation of IL-6 gingipains, lysine- and arginine-specific proteinases from Porphyromonas
gingivalis. Biochem Biophys Res Commun 1999;261:598-602.
Banerjee P, Merkel GJ, Bhunia AK. Lactobacillus delbrueckii ssp. bulgaricus B-30892 can
inhibit cytotoxic effects and adhesion of pathogenic Clostridium difficile to Caco-2 cells. Gut Pathog
2009;1:8.
Beighton D. The complex oral microflora of high-risk individuals and groups and its role in the
caries process. Community Dent Oral Epidemiol 2005;33:248255.
Belli WA, Marquis RE. Adaptation of Streptococcus mutans and Enterococcus hirae to acid
stress in continuous culture. Appl Environ Microbiol 1991; 57: 11341138.
Bernardeau M, Venoux JP, Henri-Dubernet S, Gueguen M. Safety assessment of dairy
microorganisms: The Lactobacillus genus. Int J Food Microbiol 2008;126:278-285.
Biyiko lu B, Buduneli N, Karde ler L, Aksu K, Pitkala M, Sorsa T. Gingival crevicular fluid
MMP-8 and -13 and TIMP-1 levels in patients with rheumatoid arthritis and inflammatory periodontal
disease. J Periodontol 2009;80:1307-1314.
Blanc B, Laloi P, Atlan D, Gilbert C, Portalier R. Two cell wall-associated aminopeptidases
from Lactobacillus helveticus and the purification and characterization of APII from strain ITGLl. J
Gen Microbiol 1993;139:1441-1448.
`Bob` ten Cate JM. The need for antibacterial approaches to improve caries control. Adv Dent
Res 2009;21:8-12
Bodet C, Chandad F, Grenier D. Modulation of cytokine production by Porphyromonas
gingivalis in a macrophage and epithelial cell co-culture model. Microbes Infect 2005;7:448-456.
Bodet C, Andrian E, Tanabe S, Grenier D. Actinobacillus actinomycetemcomitans
lipopolysaccharide regulates matrix metalloproteinase, tissue inhibitors of matrix metalloproteinase,
and plasminogen activator production by human gingival fibroblasts: a potential role in connective
tissue destruction. J Cell Physiol 2007;212:189-194.
61
Boekhorst J, Wels M, Kleerebzem M, Siezen RJ. The predicted secretome of Lactobacillus

plantarum WCFS1 sheds light on interactions with its environment. Microbiology 2006;152:31753183.
Borrell LN, Papapanou PN. Analytical epidemiology of periodontitis. J Clin Periodontol 2005;
32:132-158.
Bosch JA, Turkenburg M, Nazmi K, Veerman EC, de Geus EJ, Nieuw Amerongen AV. Stress
as a determinant of saliva-mediated adherence and coadherence of oral and nonoral microorganisms.
Psychosom Med 2003;65:604-612.
Braat H, Rottiers P, Hommes DW, Huyghebaert N, Remaut E, Remon JP, van Deventer SJH,
Neirynck S, Peppelenbosch M, Steidler L. A phase I trial with transgenic bacteria expressing
Interleukin-10 in Crohn`s disease. Clin Gastroenterol Hepatol 2006; 4:754-759.
Bradshaw DJ, Marsh PD. Analysis of pH-driven disruption of oral microbial communities in
vitro. Caries Res 1998;32:456-462.
Brukhardt A, Maerker R (1981). A color atlas of oral cancers. Wolf Medical Publications,
London.
Bu HF, Wang X, Zhu YQ, Williams RY, Hsueh W, Zheng H, Rozenfeld RA, Zuo XL, Tan XD.
Lysozyme-modified probiotic components protect rats against polymicrobial sepsis: role of
macrophages and cathelicidin-related innate immunity. J Immunol 2006; 177: 8767 8776.
Burne RA, Quivey RG, Marquis RE. Physiologic homeostasis and stress responses in oral
biofilms. Meth Enzymol 1999;310:441-460.
Burton JP, Chilcott CN, Moore CJ, Speiser G, Tagg JR. A preliminary study of the effect of
probiotic Streptococcus salivarius K12 on oral malodour parameters. J Appl Microbiol 2006;100:754764.
Busscher HJ, Mulder A, van der Mei HC. In vitro adhesion to enamel and in vivo colonization
of tooth surfaces by lactobacilli from bio-yoghurt. Caries Res 1999;33:403-404.
Cabrera CC, Hakeberg M, Ahlqwist M, Wedel H, Bjrklund C, Bengtsson C, Lissner L. Can
the relation between tooth loss and chronic disease be explained by socio-economic status? A 24 year
follow-up from the population study of women in Gothenburg, Sweden. Eur J Epidemiol 2005;20:229
236.
aglar E, Kargul B, Tanboga I. Bacteriotherpay and probiotics` role on oral health. Oral Dis
2005;11:131-137.
aglar E, Cildir SK, Ergeneli S, Sandalli N, Twetmen S. Salivary mutans streptococci and
lactobacilli levels after ingestion of the probiotic bacterium Lactobacillus reuteri ATCC 55730 by
straws or tablets. Acta Odontol Scand 2006;64:314-318.
aglar E, Kavaloglu SC, Kuscu OO, Sandalli N, Holgerson PL, Twetman S. Effect of chewing
gums containing xylitol or probiotic bacteria on salivary mutans streptococci and lactobacilli. Clin Oral
Investig 2007;11:425-429.
62
aglar E, Kusku OO, Cildir SK, Kuvvetli SS, Sandalli N. A probiotic lozenge administered
medical device and its effect on salivary mutans streptococci and lactobacilli. Int J Paediatr Dent
2008;18:35-39.
aglar E, Sandalli N, Twetman S, Kavaloglu S, Ergeneli S, Selvi S. Effect of yogurt with
Bifidobacterium DN-173 010 on salivary mutans streptococci and lactobacilli in young adults. Acta
Odontol Scand 2005;63:317-320.
Calkins CC, Platt K, Potempa J, Travis J. Inactivation of tumor necrosis factor-alpha by
proteinases (ginigipains) from the periodontal pathogen, Porphyromonas gingivalis. Implications of
immune evasion. J Biol Chem 1998;273: 6611-6614.
Carbo ML, Braber AF, Koenraad PM. Apparent antifungal activity of cervical lactic acid
bacteria against Penicillinum discolor is due to acetic acid in the medium. J Food Prot 2002;65:13091316.
Carvalho JC, Figueiredo MJ, Vieira EO, Mestrinho HD. Caries trends in Brazilian nonprivileged preschool children in 1996 and 2006. Caries Res 2009;43:2-9.
Caufield PW. Dental caries: an infectious and transmissible disease where have we been and
where are we going? N Y State Dent J 2005;71:23-27.
Cildir SK, Germec D, Sandalli N, Ozdemir FI, Arun T, Twetmen S, aglar E. Reduction of
salivary mutans streptococci in orthodontic patients during daily consumption of yoghurt containing
probiotic bacteria. Eur J Orthod 2009; 31:407-411.
Coeuret V, Gueguen M, Vernoux JD. In vitro screening of potential probiotic activities of
selected lactobacilli isolated from unpasteurized milk products for incorporation into soft cheese. J
Dairy Res 2004;71:451-460.
Cosseau C, Devine DA, Dullaghan E, Gardy JL, Chikatamarla A, Gellatly S, Yu LL, Pistolic J,
Falsafi R, Tagg J, Hancock RE. The commensal Streptococcus salivarius K12 downregulates the
innate immune responses of human epithelial cells and promotes host-microbe homeostasis. Infect
Immun 2008;76:4163-4175.
Darveau RP, Belton CM, Reife RA, Lamont RJ. Local chemokine paralysis, a novel pathogenic
mechanism for Porphyromonas gingivalis. Infect Immun 1998;66:1660-1665.
Davis JG, Carr JG, Cutting CV and Whiting GC (eds) (1975). The microbiology of yogurt.
Lactic Acid Bacteria in Beverages and Food. Academic Press, London.
de Vrese M, Winkler P, Rautenberg P, Harder T, Noah C, Laue C, Ott S, Hampe J, Schreiber S,
Heller K, Schrezenmeir J. Effect of Lactobacillus gasseri PA 16/8, Bifidobacterium longum SP 07/3,
B. bifidum MF 20/5 on common cold episodes: a double blind, randomized, controlled trial. Clin Nutr
2005;24:481-491.
De Vuyst L, Vandamme EJ (1994). In: Bacteriocins of lactic acid bacteria; Antimicrobial
potential of lactic acid bacteria. Blackie Academic and Professional, Glasgow, UK.
del Campo R, Bravo D, Cnton R, Ruiz-Garbajosa P, Garcia-Albiach R, Montesi-Libois A, Yste
F-J, Abraria V, Baquero F. Scarce evidence of yogurt lactic acid bacteria in human feces after dairy
yogurt consumption by healthy volunteers. Appl Environ Microbiol 2005;71:547-549.
63
Dellaglio F, Felis G; Tannock GW (eds) (2005) Taxonomy of lactobacilli and bifidobacteria. In:
Probiotics and prebiotics: scientific aspects. Caister Academic Press, Norfolk, UK.
Delley M, Germont J-E. Differentiation of Lactobacillus helveticus, Lactobacillus delbrueckii
subsp bulgaricus, subsp lactis and subsp delbrueckii using physiological and genetic tools and
reclassification of some strains from the ATCC Collection. Syst Appl Microbiol 2002;25:228-231.
DeSimone C, Vesely R, Baldinelli L, Lucci L. The adjuvant effect of yogurt on the production
of gamma-interferon by Con A-stimulated human peripheral blood lymphocytes. Nutr Rep Int
1986;33:419-433.
DeStefano F, Anda RF, Kahn HS, Williamson DF, Russel CM. Dental disease and risk of
coronary heart disease and mortality. Br Med J 1993;306:688691.
Devine DA, Cosseau C. Host defense peptides in the oral cavity. Adv Appl Microbiol 2008;63:
281-322.
Devine DA. Antimicrobial peptides in defense of the oral and respiratory tracts. Mol Immunol
2003;40:431-443.
Dewit O, Pochart P, Desjeux JF. Breath hydrogen concentration and plasma glucose, insulin
and free fatty acid levels after lactose, milk, freash or heated yogurt ingestion by healthy young adults
with or without lactose malabsorption. Nutrition 1988;4:131-135.
Dixon DR, Reife RA, Cebra JJ, Darveau RP. Commensal bacteria influence innate status within
gingival tissues: a pilot study. J Periodontol 2004;75:1486-1492.
Dogi CA, Galdeano CM, Perdigon G. Gut immune stimulation by non pathogenic Gram(+) and
Gram(-) bacteria. Comparison with a probiotic strain. Cytokine 2008;41:223-231.
Drago L, Gismondo MR, Lombardi A, de Han C, Gozzini L. Inhibition of in vitro growth of
enteropathogens by new Lactobacillus isolates of human intestinal origin. FEMS Microbiol Lett
1997;153:455-463.
Dunne C, Murphy L, Flynn S, O`Mahony L, O`Halloran S, Feeney M, Morrissey D, Thornton
G, Fitzgerald G, Daly C, Kiely B, Quigley EM, O`Sullivan GC, Shanahan F, Collins JK. Probiotics:
from myth to reality. Demonstration of functionality in animal models of disease and in human clinical
trials. Antonie Van Leeuwenhoek 1999;76:279-292.
Eberhard J, Pietschmann R, Falk W, Jepsen S, Dommisch H. The immune response of oral
epithelial cells induced by single-species and complex naturally formed biofilms. Oral Microbiol
Immunol 2009;24:325-330.
Eckmann L, Smith JR, Housley MP, Dwinell MB, Kagnoff MF. Analysis by high density
cDNA arrays of altered gene expression in human intestinal epithelial cells in response to infection
with the invasive enteric bacteria Salmonella. J Biol Chem 2000; 275: 14084-14094.
Edgar WM. Sugar substitutes, chewing gum and dental caries a review. Br Dent J
1998;184:29-32.
Ejeil A-L, Indondjo-Tchen S, Ghomrasseni S, Pellat B, Godeau G, Gogly B. Expression of
matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in healthy and
diseased human gingiva. J Periodontol 2003;74:188195.
64
Elahi S, Pang G, Ashman R, Clancy R. Enhanced clearance of Candida albicans from the oral
cavities of mice following oral administration of Lactobacillus acidophilus. Clin Exp Immunol
2005;141:29-36.
Elli M, Callegari ML, Ferrari S, Bessi E, Cattivelli D, Soldi S, Morelli L, Feuillerat NC,
Antoine JM. Survival of yogurt bacteria in human gut. Appl Environ Microb 2006;72:5113-5117.
El-Nezami HS, Polychronaki NN, Ma J, Zhu H, Ling W, Salminen EK, Juvonen RO, Salminen
SJ, Poussa T, Mykknen HM. Probiotic supplementation reduces a biomarker for increased risk of
liver cancer in young men from Southern China. Am J Clin Nutr 2006;83:1199-1203.
Elo S, Saxelin M, Salminen S. Attachment of Lactobacillus casei strain GG to human colon
caricinoma cell line Caco-2: comparison with other dairy strains. Lett Appl Microbiol 1991;13:154156.
Fabian E, Elmadfa I. Influence of daily consumption of probiotic and conventional yoghurt on
the plasma lipid profile in young healthy women. Ann Nutr Metab 2006;50:387-393.
Fernandez MF, Boris S, Barbes C. Probiotic properties of human lactobacilli strains to be used
in the gastrointestinal tract. J Appl Microbiol 2003:94:449455.
Ferrazzano GF, Amato I, Ingenito A, De Natale A, Pollio A. Anti-cariogenic effects of
polyphenols from plant stimulant beverages (cocoa, coffee, tea). Fitoterapia 2009;80:255-262.
Formanek M, Knerer B, Kornfehl J. Cytokine expression of human oral keratinocytes. ORL
1999;61:103-107.
Fujimori S, Gudis K, Mitsui K, Seo T, Yonezawa M, Tanaka S, Tatsuguchi A, Sakamoto C. A
randomized controlled trial on the efficacy of synbiotic versus probiotic or prebiotic treatment to
improve the quality of life in patients with ulcerative colitis. Nutrition 2009;25:520-525.
Fuller R. Probiotics in man and animals. J Appl Bacteriol 1989;66:365-378.
Galan JE, Zhou D. Striking a balance: modulation of the actin cytoskeleton by Salmonella. Proc
Natl Acad Sci U S A 2000; 97: 8754-8761.
Garcia RI, Krall EA, Vokonas PS. Periodontal disease and mortality from all causes in the VA
Dental Longitudinal Study. Ann Periodontol 1998;3:339349.
Garcia Vilela E, De Lourdes De Abreu Ferrari M, Oswaldo Da Gama Torres H, Guerra Pinto A,
Carolina Carneiro Aguirre A, Paiva Martins F, Marcos Andrade Goulart E, Sales Da Cunha A.
Influence of Saccharomyces boulardii on the intestinal permeability of patients with Crohn's disease in
remission. Scand J Gastroenterol 2008;43:842-848.
Garcia-Albiach R, Jose M, de Felipe P, Angulo S, Morosini M-I, Bravo D, Baquero F, del
Campo R. Molecular analysis of yogurt containing Lactobacillus delbrueckii subsp. bulgaricus and
Streptococcus thermophilus in human intestinal microbiota. Am J Clin Nutr 2008;87:91-96.
Gardner MS, Rowland MD, Siu AY, Bundy JL, Wagener DK, Stephenson JL. Comprehensive
defensin assay for saliva. Anal Chem 2009;81:557-566.
Germaine GR, Tellefson LM. Potential role of lysozyme in bactericidal activity of in vitroacquired salivary pellicle against Streptococcus faecium 9790. Infect Immun 1986;54:846-854.
65
Gibbons RJ, Etherden I. Enzymatic modification of bacterial receptors on saliva-treated

hydroxyapatite surfaces. Infect Immun 1982;36:5258.
Gilliland SE, Kim HS. Effect of viable starter culture bacteria in yogurt on lactose utilization in
humans. J Dairy Sci 1984;67:1-6.
Gionchetti P, Rizzello F, Morselli C, Poggioli G, Tambasco R, Calabrese C, Brigidi P, Vitali B,
Straforini G, Campieri M. High-dose probiotics for the treatment of active pouchitis. Dis Colon
Rectum. 2007;50:2075-2082.
Giraffa G, Bossi MG, Fornasari E. Bacteriocin production by Lactobacillus delbrueckii subsp.
lactis strains. Microbiol Aliments Nutrition 1989;7:139-143.
Giraffa G, Lazzi C, Gatti M, Rossetti L, Mora D, Neviani E. Molecular typing of Lactobacillus
delbrueckii of dairy origin by PCR-RFLP of protein-coding genes. Int J Food Microbiol 2003;82:163172.
Girginoff T. Microbiology of the Animal Products. State Publishing House for Agriculture
Literature, 1959, Sofia, Bulgaria, p. 52.
Grafstrm R; Freshney IR (eds) (2002). Culture of epithelial cells; Structure of oral mucosa.
John Wiley & Sons, 2nd ed., New York, USA.
Grayson R, Douglas CW, Heath J, Rawlinson A, Evans GS. Activation of human matrix
metalloproteinase-2 by gingival crevicular fluid and Porphyromonas gingivalis. J Clin Periodontol
2003;30:542-550.
Grschl M, Wendler O, Topf HG, Bohlender J, Khler H. Significance of salivary
adrenomedullin in the maintenance of oral health: Stimulation of oral cell proliferation and
antibacterial properties. Regul Pept 2009;154:16-22.
Grudianov AI, Dmitrieva NA, Fomenko EV. Use of probiotics Bifidumbacterin and Acilact in
tablets in therapy of periodontal inflammations. Stomatologiia (Mosk) 2002;81:39-43.\
Gu Y, Lee HM, Sorsa T, Simon SR, Golub LM. Doxycyline inhibits mononuclear cell-mediated
connective tissue breakdown. FEMS Immunol Med Microbiol 2009; Nov 10.
Guan SM, Shu L, Fu SM, Liu B, Xu XL, Wu JZ. Prevotella intermedia induces matrix
metalloproteinase-9 expression in human periodontal ligament cells. FEMS Microbiol Lett
2008;283:47-53.
Guandalini S, Pensabene L, Zikri MA, Dias JA, Casali LG, Hoekstra H, Kolacek S, Massar K,
Micetic-Turk D, Papadopoulou A, de Sousa JS, Sandhu B, Szajewska H, Weizman Z. Lactobacillus
GG administered in oral rehydration solution to children with acute diarrhea: a multicenter European
trial. J Pediatr Gastroenterol Nutr 2000;30:54-60.
Guarner F, Perdigon G, Corthier G, Salminen S, Koletzko B, Morelli L. Should yoghurt
cultures be considered probiotic? Br J Nutr 2005;93:783-786.
Gullemand E, Tondu F, Lacoin F, Schrezenmeir J. Consumption of a fermented dairy product
containing the probiotic Lactobacillus casei DN-114001 reduces the duration of respiratory
infections in the elderly in a randomised controlled trial. Brit J Nutr 2010;103:58-68.
66
Guslandi M, Giollo P, Testoni PA. A pilot trial of Saccharomyces boulardii in ulcerative colitis.
Eur J Gastroenterol Hepatol 2003; 15: 697-698.
Guslandi M, Mezzi G, Sorghi M, Testoni PA. Saccharomyces boulardii in maintenance
treatment of Crohn`s disease. Dig Dis Sci 2000; 45: 1462-1464.
Hahnel S, Rosentritt M, Handel G, Brgers R. Influence of saliva substitute films on initial
Streptococcus mutans adhesion to enamel and dental substrata. J Dent 2008;36:977-983.
Hammerman C, Bin-Nun A, Kaplan M. Safety of probiotics: comparison of two popular strains.
BMJ 2006;33:1006-1008.
Hammes, WP, Vogel RF; Wood BJB and Holzapfel WH (eds). (1995). The Genera of Lactic
Acid Bacteria; The genus Lactobacillus. Chapman & Hall, London.
Handelsman J. Metagenomics: application of genomics to uncultured microorganisms.
Microbiol Mol Biol Rev 2004;68:669-685.
Hasegawa Y, Mans JJ, Mao S, Lopez MC, Baker HV, Handfield M, Lamont RJ. Gingival
epithelial cell transcriptional responses to commensal and opportunistic oral microbial species. Infect
Immun 2007;75:2540-2547.
Hassan AN, Frank JF; . Marth EH and Steele JL (eds) (2001). Applied Dairy Microbiology;
Starter cultures and their use. Marcel Dekker, Inc, New York, USA.
Hatakka K, Savilahti E, Pnk A, Meurman JH, Poussa T, Nse L, Saxelin M, Korpela R.
Effect of long term consumption of probiotic milk on infections in children attending day care centres:
double blind, randomised trial. BMJ 2001;322:1318-1319.
Hatakka K, Ahola AJ, Yli-Knuuttila H, Richardson M, Poussa T, Meurman JH, Korpela R.
Probiotics reduce the prevalence of oral candida in the elderly--a randomized controlled trial. J Dent
Res 2007;86:125-130.
Hatakka K, Holma R, El-Nezami H, Suomalainen T, Kuisma M, Saxelin M, Poussa T,
Mykknen H, Korpela R. The influence of Lactobacillus rhamnosus LC705 together with
Propionibacterium freudenreichii ssp. shermanii JS on potentially carcinogenic bacterial activity in
human colon. Int J Food Microbiol 2008;128:406-410.
Hatakka K, Saxelin M. Probiotics in intestinal and non-intestinal infectious diseases - clinical
evidence. Curr Pharm Des 2008;14;1351-1367.
Haugejorden O, Birkeland JM. Analysis of the ups and downs of caries experience among
Norwegian children aged five years between 1997 and 2003. Acta Odontol Scand 2005;63:115-122.
Haukioja A, Sderling E, Tenovuo J. Acid production from sugars and sugar alcohols by
probiotic lactobacilli and bifidobacteria in vitro. Car Res 2008;42:449-453.
Haukioja A, Yli-Knuuttila H, Loimaranta V, Kari K, Ouwehand AC, Meurman JH, Tenovuo J.
Oral adhesion and survival of probiotic and other lactobacilli and bifidobacteria in vitro. Oral
Microbiol Immunol 2006;21:326-332.
He X, Lux R, Kuramitsu HK, Anderson MH, Shi W. Achieving probiotic effects via
modulating oral microbial ecology. Adv Dent Res 2009;21:53-56
67
Hedberg M, Hasslof P, Sjstrm I, Twetman S, Stecksen-Blicks C. Sugar fermentation in

probiotic bacteria an in vitro study. Oral Microbiol Immunol 2008;23:482-485.
Hickson M, D`Souza AL, Muthu N, Rogers T, Want S, Bulpitt CJ. Use of probiotic
Lactobacillus preparation to prevent diarrhoea associated with antibiotics: randomised double blind
placebo controlled trial. BMJ 2007;1:5.
Hillman JD, McDonell E, Cramm T, Hillman CH, Zahradnik RT. A spontaneous lactate
dehydrogenase deficient mutant of Streptococcus rattus for use as a probiotic in the prevention of
dental caries. J Appl Microbiol 2009;107;1551-1558.
Hiratsuka K, Wang B, Sato Y, Kuramitsu H. Regulation of sucrose-6-phosphate hydrolase
activity in S. mutans: characterization of scrR gene. Infect Immun 1998;66:3736-3743.
Hojo K, Nagaoka S, Ohshima T, Maeda N. Bacterial interactions in dental biofilm
development. J Dent Res 2009;88:982-990.
Holzapfel WH, Haberer P, Geisen R, Bjrkroth J, Schillinger U. Taxonomy and important
featured of probiotic microorganisms in food and nutrition. Am J Clin Nutr 2001;73:365-373.
Howey R T, Lock CM, Moore LVH. Subspecies names automatically created by rule 46. Int J
Syst Bacteriol 1990;40:317319.
Hunter CJ, Williams M, Petrosyan M, Guner Y, Mittal R, Mock D, Upperman JS, Ford HR,
Prasadarao NV.
Lactobacillus bulgaricus prevents intestinal epithelial cell injury caused by
Enterobacter sakazakii-induced nitric oxide both in vitro and in the newborn rat model of necrotizing
enterocolitis. Infect Immun 2009;77:1031-1043.
Ishikawa H, Aiba Y, Nakanishi M, Oh-Hashi Y, Koga Y. Suppression of periodontal
pathogenic bacteria in the saliva of humans by the administration of Lactobacillus salivarius TI2711. J
Jap Assoc Periodontol 2003;45:105-112.
Isolauri E, Arvola T, Sutas Y, Moilanen E, Salminen S. Probiotics in the management of atopic
eczema. Clin Exp Allergy 2000; 30:1604-1610.
Jacobsen CN, Rosenfeldt Nielsen V, Hayford AE, Moler PL, Michaelsen KF, Paerregaard A,
Sandstrm B, Tvede M, Jakobsen M. Screening of probiotic activities of forty-seven strains of
Lactobacillus spp. by in vitro techniques and evaluation of the colonization ability of five selected
strains in humans. Appl Environ Microbiol 1999;65:4949-4956.
Jansson L, Lavstedt S, Frithiof L. Relationship between oral health and mortality rate. J Clin
Periodontol 2002;29:10291034.
Jayaraman JC, Penders JE, Burne RA. Transcriptional analysis of the Streptococcus mutans
hrcA, grpE, and dnaK genes and regulation of the expression in response to heat shock and
environmental acidification. Mol Microbiol 1997;25:329-341.
Johansson E, Claesson R, van Dijken JW. Antibacterial effect of ozone on cariogenic bacterial
species. J Dent 2009;37:449-453.
Johnson LL, Dyer R, Hupe DJ. Matrix metalloproteinases. Curr Opin Chem Biol 1998;2:466
471.
68
Kagnoff MF, Eckmann L. Epithelial cells as sensors for microbial infection. J Clin Invest
1997;100: 6-10.
Kakessy DA, Piguet JD. New method for detecting bacteriocin production. J Appl Microbiol
1970;20:282-283.
Kang MS, Chung J, Kim SM, Yang KH, Oh JS. Effect of Weissella cibaria isolates on the
formation of Streptococcus mutans biofilm. Caries Res 2005;40:418-425.
Kang MS, Kim BG, Chung J, Lee HC, Oh JS. Inhibitory effect of Weissella cibaria isolates on
the production of volatile sulfur compounds. J Clin Periodontol 2006;33:226-232.
Katrandjiev K. The Bulgarian Yogurt, 1962, Publishing House of Bulgarian Academy of
Science, Bulgaria (in Bulgarian).
Kawase M, He F, Kubota A, Hiramatsu M, Saito H, Ishii T, Yasueda H, Akiyama K. Effect of
fermented milk prepared with two probiotic strains on Japanese cedar pollinosis in a double-blind
placebo-controlled clinical study. Int J Food Microbiol 2009;128:429-434.
Kazor CE, Mitchel PM, Lee AM, Stokes LN, Loesche WJ, Dewhirst FE, Paster BJ. Diversity of
bacterial populations on the tongue dorsa of patients with halitosis and healthy patients. J Clin
Microbiol 2003;41:558-563.
Keijser BJ, Zaura E, Huse SM, van der Vossen JM, Schuren FH, Montijn RC, ten Cate JM,
Crielaad VV. Pyrosequencing analysis of the oral microflora of healthy adults. J Dent Res
2008;87:1016-1020.
Kiessling G, Schneider J, Jahreis G. Long-term consumption of fermented dairy products over 6
months increases HDL cholesterol. Eur J Clin Nutr 2002;56:843-849.
Keller D, Costerton JW. Oral biofilm: entry and immune system response. Compend Contin
Educ Dent 2009;30:24-32.
Kinane DF, Mooney J, Ebersole JL. Humoral immune response to Actinobacillus
actinomycetemcomitans and Porphyromonas gingivalis in periodontal disease. Periodontol 2000
1999;20:289-340.
Kirjavainen PV, Salminen SJ, Isolauri E. Probiotic bacteria in the management of atopic
disease: underscoring the importance of viability. J Pediatr Gastroenterol Nutr 2003;36:223-227.
Kitazawa H, Watanabe H, Shimosato T, Kawai Y, Itoh T, Saito T. Immunostimulatory
oligonucleotide, CpG-like motif exists in Lactobacillus delbrueckii ssp. bulgaricus NIAI B6. Int J
Food Microbiol 2003;85:11-21.
Klaenhammer T, Altermann E, Pfeiler E, Buck BL, Goh Y-J, O`Flaherty S, Barrangou R,
Duong T. Functional genomics of probiotic lactobacilli. J Clin Gastroenterol 2008;42:160-161.
Kleeman EG, Klaenhammer T. Adherence of Lactobacillus species to human fetal intestinal
cells. J Dairy Sci 1982;65:2063-2069.
Koivunen AL, Maisi P, Konttinen YT, Prikk K, Sandholm M. Collagenolytic activity and its
sensitivity to doxycycline inhibition in tracheal aspirates of horses with chronic obstructive pulmonary
disease. Acta Vet Scand 1997;38: 916.
Kollath W. Diagnosis of intestinal dysbacteria. Zentralbl Bactriol Orig 1954;161:501-503.
69
Kll-Klais P, Mndar R, Leibur E, Marcotte H, Hammarstm L, Mikelsaar M. Oral lactobacilli

in chronic periodontitis and periodontal health: species composition and antimicrobial activity. Oral
Krisanaprakornkit S, Kimball JR, Weinberg A, Darveau PR, Bainbridge BW, Dale BA.
Inducible expression of human beta-defensin 2 by Fusobacterium nucleatum in oral epithelial cells:
multiple signaling pathways and role of commensal bacteria in innate immunity and the epithelial
barrier. Infect Immun 2000;65:2907-2915.
Kruis W, Fric P, Pokrotnieks J, Fixa B, Kascak M, Kamm MA, Weismueller J, Beglinger C,
Stolte M, Wolff C, Schulze J. Maintaining remission of ulcerative colitis with the probiotic
Escherichia coli Nissle 1917 is as effective as with standard mesalazine. Gut 2004;53:1617-1623.
Kruis W. Maintenance of remission in ulcerative colitis is equally effective with Escherichia
coli Nissle 1917 and with standard mesalamine. Dig Dis Week 2001, abstract 680.
Kuramitsu HK. Molecular genetic analysis of the virulence of oral bacterial pathogens: an
historical perspective. Crit Rev Oral Biol Med 2003;14:331344.
Kuitunen M, Kukkonen K, Juntunen-Backman K, Korpela R, Poussa T, Tuure T, Haahtela T,
Savilahti E. Probiotics prevent IgE-associated allergy until age 5 years in cesarean-delivered children
but not in the total cohort. J Allergy Clin Immunol 2009;123:335-341.
Lacobsen CN, Rosenfeldt NV, Hayford AE, Moller PL, Michaelsen KF, Paerregeerd A,
Sandstrom B, Tvede M, Jakobsen M. Screening of probiotic activities of forty-seven strains of
Lactobacillus spp. by in vitro techniques and evaluation of the colonization ability of five selected
strains in humans. Appl Environ Microbiol 1999;65:29-38.
Laloi P, Atlan D, Blanc B, Gilbert C, Portalier R. Cell wall associated proteinase of
Lactobacillus delbrueckii subsp. bulgaricus CNRZ397: differential extraction, purification and
properties of the enzyme. Appl Microbiol Biothechnol 1991;136:196-204.
Land MH, Rouster-Stevensen K, Woods CR, Cannon ML, Cnota J, Shetty AK. Lactobacillus
sepsis associated with probiotic therapy. Pediatrics 2005;115:178-181.
Lee C. Changes in n-Hexanal content of peanut milk fermented with lactic acid bacteria. Food
Sci Biotechnol 2001;10:387390.
Lee YK, Wong SF; Salminen S, von A Wright (Eds) (1993). Lactic acid bacteria : Stability of
lactic acid bacteria in fermented milk. Marcel Dekker. New York, USA.
Lee YK, Selection and maintenance of probiotic microorganisms. Lee YK, Salminen S (eds)
(2009). In: Handbook of probiotics and prebiotics, John Wiley & Sons, Inc., Hoboken, New Jersey,
USA, pp.178-185.
Lerebours E, N`Djitoyap NC, Lavoine A, Hellot MF, Antonie JM, Colin R. Yogurt and
fermented-then-pasteurized milk: effects of short-term and long-term ingestion on lactose absorption
and mucosal lactase activity in lactase-deficient subjects. Am J Clin Nutr 1989;49:823-827.
Leyer GJ, Li S, Mubasher ME, Reifer C, Ouwehand AC. Probiotic effects on cold and
influenza-like symptom incidence and duration in children. Pediatrics 2009;124:172-179.
70
Li Y-H, Burne RA. Regulation of the gtfBC and ftf genes of Streptococcus mutans in biofilms
in response to pH and carbohydrate. Microbiology 2001; 147: 28412848.
Li Y-H, Lau PC, Lee JH, Ellen RP, Cvitkovitch DG. Natural genetic transformation of
Streptococcus mutans growing in biofilms. J Bacteriol 2001a; 183: 897908.
Li Y-H, Hanna MN, Svensa ter G, Ellen RP, Cvitkovitch DG. Cell density modulates acid
adaptation in Streptococcus mutans: implications for survival in biofilms. J Bacteriol 2001b;183:6875
6884.
Li Y-H, Tang N, Aspiras M B, Lau P C Y, Lee J H, Ellen R P Cvitkovitch DG. A quorum
sensing signaling system essential for genetic competence in Streptococcus mutans is involved in
biofilm formation. J Bacteriol 2002;184: 26992708.
Lick S, Drescher K, Heller K. Survival of Lactobacillus delbrueckii subsp. bulgaricus and
Streptococcus thermophilus in the terminal ileum of fistulated Gttingen minipigs. Appl Environ
Microbiol 2001;67:4137-4143.
Lilly DM, Stillwell RH. Probiotics: growth-promoting factors produced by microorganisms.
Science 1965;147:747-748.
Lima LM, Motisuki C, Spolodorio DMP, Santos-Pinto L. In vitro evaluation of probiotics
microorganisms adhesion to an artificial caries model. Europ J Clin Nutr 2005;59:884-886.
Liong MT. Safety of probiotics: translocation and infection. Nutr Rev 2008;66:192-202.
Loesche WJ. Role of Streptococcus mutans in human dental decay. Microbiol Rev
1986;50:353-380.
Loesche WJ. The specific plaque hypothesis and the antimicrobial treatment of periodontal
disease. Dent Update 1992;19:6874
Loessner M, Guenther S, Steffan S, Scherer S. A pedocin-producing Lactobacillus plantarum in
a multispecies cheese surface microbial ripening consortium. Appl Environ Microbiol 2003;69:18541857.
Lorca G, Torino MI, Font de Valdez G, Ljungh A A. Lactobacilli express cell surface proteins
which mediate binding of immobilized collagen and fibronectin. FEMS Microbiol Lett 2002;206:3137.
Majamaa H, Isolauri E. Probiotics: a novel approach in the management of food allergy. J
Allergy Clin Immunol 1997;99:179-185.
Makino S, Ikegami S, Kano H, Sashihara T, Sugano H, Horiuchi H, Saito T, Oda M.
Immunomodulatory effects of polysaccharides produced by Lactobacillus delbrueckii supsp.
bulgaricus OLL1073-R1. J Dairy Sci 2006;89:2873-2881.
Mantzourani M, Gilbert SC, Sulong HNH, Sheehy EC, Tank S, Fenlon M, Beighton D. The
isolation of bifidobacteria from occlusal carious lesions in children and adults. Caries Res
2009;43:308-313
Marcaccini AM, Novaes AB Jr, Meschiari CA, Souza SL, Palioto DB, Sorgi CA, Faccioli LH,
Tanus-Santos JE, Gerlach RF. Circulating matrix metalloproteinase-8 (MMP-8) and MMP-9 are
71
increased in chronic periodontal disease and decrease after non-surgical periodontal therapy. Clin
Chim Acta 2009;409:117-122.
Marin
ML,
Tejada-Simon
MV,
Lee
JH,
Murtha
J,
Ustunol
Z,
Pestka
JJ.
Stimulation of cytokine production in clonal macrophage and T-cell models by Streptococcus

thermophilus: comparison with Bifidobacterium sp. and Lactobacillus bulgaricus. J Food Prot
1998;61:859-864.
Markoff VL. Applied Microbiology 1925, PELIN, Sofia, Bulgaria (in Bulgarian).
Marsh PD. Microbial ecology of dental plaque and its significance in health and disease. Adv
Dent Res 1994;8:263-271.
Marsh PD. Dental plaque: biological significance of a biofilm and community life-style. J Clin
Periodontol 2005;32:7-15.
Marteau P, Lemann M, Seksik P, Laharie D, Colombel JF, Bouhnik Y, Cadiot G, Soule JC,
Bourreille A, Metman E, Lerebours E, Carbonnel F, Dupas JL, Veyrac M, Coffin B, Moreau J, Abitbol
V, Blum-Sperisen S, Mary JY. Ineffectiveness of Lactobacillus johnsonii LA1 for prophylaxis of
postoperative recurrence in Crohn`s disease: a randomised, double-blind, placebo-controlled GETAID
trial. Gut 2006;55: 842-847.
Marteau P, Minekus M, Havenaar R, Huis JHJ. Survival of lactic acid bacteria in a dynamic
model of the stomach and small intestine: validation and the effects of bile. J Dairy Sci 1997;80:10311037.
Marteau PR. Probiotics in clinical conditions. Clin Rev Allergy Immunol 2002;22:255-273.
Martin FE, Nadkarni MA, Jaques NA, Hunter H. Quantitative microbiological study of human
carious dentine by culture and real-time PCR: association of anaerobes with histopathological changes
in chronic pulpitis. J Clin Microbiol 2002;40:1698-1704.
Mater DDG, Bretigny L, Firmesse O, Flores M-J, Mogenet A, Bresson J-L, Corthier G.
Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus survive gastrointestinal
transit of healthy volunteers consuming yogurt. FEMS Microbiol Lett 2005;250:185-187.
Matsumoto M, Tsuji M, Sasaki H, Fujita K, Nomura R, Nakano K, Shintani S, Ooshima T.
Cariogenicity of the probiotic bacterium Lactobacillus salivarius in rats. Caries Res 2005;39:479-483.
Matsuoka T, Sugano N, Takigawa S, Takane M, Yoshimura N, Ito K, Koga Y. Effect of oral
Lactobacillus salivarius TI 2711 (LS1) administration on periodontopathic bacteria in subgingival
plaque. J Jap Assoc Periodontol 2006;48:315-324.
McAuliffe O, Ross RP, Hill C. Lantibiotics: structure, biosynthesis and mode of action. FEMS
Microbiol Rev 2001;25:285-308.
Medina M, Izquierdo E, Ennahar S, Sanz Y. Differential immunomodulatory properties of
Bifidobacterium logum strains: relevance to probiotic selection and clinical applications. Clin Exp
Immunol 2007;150:531-538.
Mercier J, Lindow SE. Role of leaf surface sugars in colonization of plants by bacterial
epiphytes. Appl Environ Microbiol 2000;66:369374.
72
Meurman JH. Probiotics: do they have a role in oral medicine and dentistry? Eur J Oral Sci
2005;113:188-196.
Meurman JH; Charalampopoulos D and Rastall R A. (Eds) (2009). : Prebiotics and Probiotics:
Science and technology; Prebiotics and Probiotics and Oral Health. Springer and Business media,
Germany.
Meydani S, Ha WK. Immunologic effect of yogurt. Am J Clin Nutr 2000; 71: 861872.
Michaylova M, Dimitrov Z, Isawa K, Minkova Sv. Isolation and characterization of L.
bulgaricus and S. thermophilus from plants and home-made yogurts. Abstract of International
Symposium on Original Bulgarian Yogurt. Sofia, Bulgaria, 2527 May, 2005, pp. 1112, (in
Bulgarian).
Miele E, Pascarella F, Giannetti E, Quaglietta L, Baldassano RN, Staiano A. Effect of a
probiotic preparation (VSL#3) on induction and maintenance of remission in children with ulcerative
colitis. Am J Gastroenterol 2009;104:437-443.
Mikelsaar M, Turi M, Lenzner H, Kolts K, Kirch R, Lenzner A. Interactions between mucosal
and luminal microflora of gastrointestine. Die Nahrung 1987;31:449-456.
Millar MR, Bacon C, Smith SL, Walker V, Hall MA. Enteral feeding of premature infants with
Lactobacillus GG. Arch Dis Child 1993;69:483-487.
Milliere JB, Abidi FZ, Lefebvre G. Taxonomic characterization of Lactobacillus delbrueckii
subsp. bulgaricus isolates from a Cameroonian zebus fermented raw milk. J Appl Bacteriol
1996;80:583588.
Mimura T, Rizzello F, Helwig U, Poggioli G, Schreiber S, Talbot IC, Nicholls RJ, Gionchetti P,
Kampieri M, Kamm MA. Once daily high dose probiotic therapy (VSL#3) for maintaining remission
in recurrent or refractory pouchitis. Gut 2004;53:108-114.
Miteva V, Ivanova I, Budakov I, Pantev A, Stefanova T, Danova S, Moncheva P, Mitev V,
Dousset X, Boyaval P. Detection and characterization of a novel antibacterial substance produced by a
Lactobacillus delbrueckii subsp. bulgaricus strain 1043. J Appl Microbiol 1998;85:603-614.
Modesto A, Drake DR. Multiple exposures to chlorhexidine and xylitol: adhesion and biofilm
formation by Streptococcus mutans. Curr Microbiol 2006;52:418-423.
Montalto M, Vastola M, Marigo L, Covino M, Graziosetto R, Curigliano V, Santoro L, Cuoco
L, Manna R, Gasbarrini G. Probiotic treatment increases salivary counts of lactobacilli: a double-blind,
randomized, controlled study. Digestion 2004;69:53-56.
Montes RG, Bayless TM, Saavedra JM, Perman JA. Effect of milks inoculated with
Lactobacillus acidophilus or a yogurt starter culture in lactose-maldigesting children. J Dairy Sci
1995;78:1657-1664.
Morency H, Mota-Meira M, LaPointe G, Lacroix C, Lavoie MC. Comparison of the activity
spectra against pathogens of bacterial strains producing a mutacin or a lantibiotic. Can J Microbiol
2001;47:322-331.
73
Morita I, Nakagaki H, Kato K, Murakami T, Tsuboi S, Hayashizaki J, Toyama A, Hashimoto

M, Simozato T, Morishita N, Kawanaga T, Igo J, Sheiham A. Relationship between survival rates and
number of natural teeth in an elderly Japanese population. Gerodontology 2006;23:214218.
Mychailova M, Minkova S, Kimura K, Sasaki T, Isawa K. Isolation and characterization of
Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus from plants in Bulgaria.
FEMS Microbiol Lett 2007;269:160-169.
Nackaerts O, Jacobs R, Quirynen M, Rober M, Sun T, Teughels W. Replcement therapy for
periodontitis: a pilot radiographic evaluation in a dog model. J Clin Periodontol 2008;35:1048-1052.
Nagafuchi S, Takahashi T, Yajima T, Kuwata T, Hirayama K, Itoh K. Strain dependency of the
immunopotentiating activity of Lactobacillus delbrueckii subsp. bulgaricus. Biosci Biotechnol
Biochem 1999;63:474-479.
Nagase H, Enghild JJ, Suzuki K, Salvesen G. Stepwise activation mechanisms of the precursor
of matrix metalloproteinase-3 (stromelysisn) by proteinases and (4-aminophenyl)mercuric acetate.
Biochemistry 1990;29:57835789.
Naito S, Koga H, Yamaguchi A, Fujimoto N, Hasui Y, Kuramoto H, Iguchi A, Kinukawa N.
Prevention of recurrence with epirubicin and lactobacillus casei after transurethral resection of bladder
cancer. J Urol 2008;179:485-490.
Nakhjiri SF, Park Y, Yilmaz O, Chung WO, Watanabe K, El-Sabaeny A, Park K, Lamont RJ.
Inhibition of epithelial cell apoptosis by Porphyromonas gingivalis. FEMS Microbiol Lett
2001;200:145-149.
Nse L, Hatakka K, Savilahti E, Saxelin M, Pnk A, Poussa T, Korpela R, Meurman JH.
Effect of long-term consumption of a probiotic bacterium, Lactobacillus rhamnosus GG, in milk on
dental caries and caries risk in children. Caries Res 2001;35:412-420.
Nettles CG, Barefoot SF. Biochemical and genetic characteristics of bacteriocins of foodassociated lactic acid bacteria. J Food Prot 1993; 56: 338356.
Nicolas P, Bessieres P, Ehrlich DS, Maguin E, van de Guchte M. Extensive horizontal transfer
of core genome genes between two Lactobacillus species found in the gastrointestinal tract. BMC
Evoluutionary Biol 2007;7:141.
Norbert W, Shhillinger U, Kandler O. Lactobacillus lactis, Lactobacillus leichmannii and
Lactobacillus bulgaricus, Subjective Synonyms of Lactobacillus delbrueckii, and description of
Lactobacillus delbrueckii ssp. lactis comb. nov. and Lactobacillus delbrueckii ssp. bulgaricus comb.
nov. Syst Appl Microbiol 1983;4: 552557.
Nrskov-Lauritsen N, Kilian M. Reclassification of Actinobacillus actinomycetemcomitans,
Haemophilus aphrophilus, Haemophilus paraphrophilus and Haemophilus segnis as Aggregatibacter
actinomycetemcomitans gen. nov., comb. nov., Aggregatibacter aphrophilus comb. nov. and
Aggregatibacter segnis comb. nov., and emended description of Aggregatibacter aphrophilus to
include V factor-dependent and V factor-independent isolates. Int J Syst Evol Microbiol
2006;56:2135-2146.
74
Oberhelman RA, Gilman RH, Sheen P, Taylor DN, Black RE, Cabrera L, Lescano AG, Meza
R, Madico G. A placebo-controlled trial of Lactobacillus GG to prevent diarrhea in undernourished
Peruvian children. J Pediatr 1999;134:15-20.
Okamoto T, Akaike T, Suga M, Tanase S, Horie H, Miyajima S, Ando M, Ichinose Y, Maeda
H. Activation of human matrix metalloproteinases by various bacterial proteinases. J Biol Chem
1997;272:60596066.
Okamoto T, Akuta T, Tamura F, van Der Vliet A, Akaika T. Molecular mechanism for
activation and regulation of matrix metalloproteinases during bacterial infections and respiratory
inflammation. Biol Chem 2004;385:9971006.
Orla-Jensen S (1919). The lactic acid bacteria. Host., Copenhagen, Denmark.
Ortiz-Andrellucchi A, Snchez-Villegas A, Rodrguez-Gallego C, Lemes A, Molero T, Soria A,
Pea-Quintana L, Santana M, Ramrez O, Garca J, Cabrera F, Cobo J, Serra-Majem L.
Immunomodulatory effects of the intake of fermented milk with Lactobacillus casei DN114001 in
lactating mothers and their children. Br J Nutr 2008;100:834-845.
Ostengo MC, Nader-Macias EM. Hydroxylapatite beads as an experimental model to study
adhesion of lactic acid bacteria from the oral cavity to hard tissues. Methods Mol Biol 2004;268:447452.
sterberg T, Mellstrm D, Sundh V. Dental health and functional ageing. A study of 70-yearold people. Community Dent Oral Epidemiol 1990;18:313318.
Ott A, Fay LB, Chaintreau A. Determination and origin of the aroma impact compounds of
yogurt flavour. J Agr Food Chem 1997; 45: 850858.
Ouwehand A, Isolauri E, Kirjavainen PV, Tlkk S, Salminen SJ. The mucus binding of
Bifidobacterium lactis Bb12 is enhanced in the presence of Lactobacillus GG and Lact. delbrueckii
subsp. bulgaricus. Lett Appl Microbiol 2000;30:10-13.
Ouwehand AC, Btsman A, Salminen S. Probiotics for the skin: a new area of potential
application? Lett Appl Microb 2003; 36: 327331.
Ouwehand AC, Nermes M, Collado MC, Rautonen N, Salminen S, Isolauri E. Specific
probiotics alleviate allergic rhinitis during the birch pollen season. World J Gastroenterol
2009;15:3261-3268.
Ouwehand AC, Tuomola EM, Tlkk S, Salminen S. Assessment of adhesion properties of
novel probiotic strains to human intestinal mucus. Int J Food Microbiol 2001;64:119126.
Parker RB. Probiotics: The other half of the antibiotics story. Animal Nutr Hlth 1974;29:4-8.
Paster BJ, Olsen I, Aas JA, Dewhirst FE. The breadth of bacterial diversity in the human
periodontal pocket and other oral sites. Periodontol 2000 2006;42:80-87.
Pattamapun K, Tiranathanagul S, Yongchaitrakul T, Kuwatanasuchat J, Pavasant P. Activation
of MMP-2 by Porphyromonas gingivalis in human periodontal ligament cells. J Periodontal Res
2003;38:115-121.
75
Pedone CA, Bernabeu AO, Postaire ER, Bouley CF, Reinert P. The effect of supplementation
with milk fermented by Lactobacillus casei (strain DN-114 001) on acute diarrhea in children
attending day care centers. Int J Clin Pract 1999;53:179-184.
Pereyra DS, Lemannier D. Induction of human cytokines by bacteria used in dairy foods.
Nutrition Res 1993;13:1127-1140.
Petry S, Furlan S, Crepeau MJ, Cerning J, Desmazeaud M. Factors affecting exocellular
polysaccharide production by Lactobacillus delbrueckii subsp. bulgaricus grown in a chemically
defined medium. Appl Environ Microbiol 2000;8:34273431.
Petti S, Tarsitani G, Simonetti S`Arca A. A randomized clinical trial of the effect of yoghurt on
the human salivary microflora. Arch Oral Biol 2001;46:705-712.
Pham LC, van Spanning RJM, Rling WFM, Prosperi AC, Terefework Z, ten Cate JM,
Crielaard W, Zaura E. Effects of probiotic Lactobacillus salivarius W24 on the compositional stability
of oral microbial communities. Arch Oral Biol 2009;54:132-137.
Pigeon RM, Cuesta EP, Gilliland SE. Binding of free bile acids by cells of yogurt starter culture
bacteria. J Dairy Sci 2002;85:2705-2710.
Pirisi L, Creek KE, Domger J, Di Paolo JA. Continuous cell lines with altered growth and
differentiation properties originate after transfection of human keratinocytes with human
papillomavirus type 16 DNA. Carcinogenesis 1998;9:1573-1579.
Pochard P, Gosset P, Grangette C, Andre C, Tonnel AB, Pestel J, Mercenier A. Lactic acid
bacteria inhibit HT2 cytokine production by mononuclear cells from allergic patients. J Allergy Clin
Immunol 2002;110:617-623.
Potempa J, Banbula A,Travis J. Role of bacterial proteinases in matrix destruction and
modulation of host responses. Periodontol 2000 2000;24:153192.
Rafter J, Bennett M, Caderni G, Clune Y, Hughes R, Karlsson PC, Klinder A, O'Riordan M,
O'Sullivan GC, Pool-Zobel B, Rechkemmer G, Roller M, Rowland I, Salvadori M, Thijs H, Van Loo J,
Watzl B, Collins JK. Dietary synbiotics reduce cancer risk factors in polypectomized and colon cancer
patients. Am J Clin Nutr 2007;85:488-496.
Reddy GV, Shahani KM, Friend BA, Chandan, RC. Natural antibiotic activity of Lactobacillus
acidophilus and bulgaricus. III Production and partial purification of bulgarican from Lactobacillus
bulgaricus. Cultured Dairy Products Journal 1984;19:711.
Reid G, Beuerman D, Heinemann C, Bruce AW. Probiotic Lactobacillus dose required to
restore and maintain a normal vaginal flora. FEMS Immunol Med Microbiol 2001;32:37-41.
Reid G, Sanders ME, Gaskins HR, Gibson GR, Mercenier A, Rastall R, Roberfroid M,
Rowland I, Cherbut C, Klaenhammer TR. New scientific paradigms for probiotics and prebiotics. J
Clin Gastroenterol 2003;37:105-118.
Reinheimer JA, Demkow MR, Cardioti MC. Inhibition of coliform bacteria by lactic acid
cultures. Austr J Dairy Technol 1990;45:59.
Rembacken BJ, Snelling AM, Hawkey PM, Dixon ATR. A double blind trial on non pathogenic
E. coli vs mesalazine for the treatment of ulcerative colitis. Gut 1997;41:3911.
76
Reynolds JJ , Meikle MC. Mechanisms of connective tissue matrix destruction in periodontitis.

Periodontol 2000 1997;14:144157.
Ricketts DN, Pitts NB. Novel Operative treatment options. Monogr Oral Sci 2009;21:174-187.
Rizkalla SW, Luo J, Kabir M, Chevalier A, Pacher N, Slama G. Chronic consumption of fresh
but not heated yogurt improves breath-hydrogen status and short-chain fatty acid profiles: a controlled
study in healthy men with or without lactose maldigestion. Am J Clin Nutr 2000;72:1474-1479.
Roberts AP, Mullany P. Genetic basis of horizontal gene transfer among oral bacteria.
Periodontol 2000 2006;42:36-46.
Rogosa M, Hansen PA. Nomenclatural considerations of certain species of Lactobacillus
Beijerinck: Request for an opinion. Int J Syst Bacteriol 1971; 21:177-186.
Rosenberg M, Gutnick D, Rosenberg E Adherence of bacteria to hydrocarbons: a simple
method for measuring cell-surface hydrophobicity. FEMS Microbiol Lett 1980;9:2933.
Rosenfeldt V, Benfeldt E, Nielsen SD, Michaelsen KF, Jeppesen DL, Valerius NH, Paerregaard
A. Effect of probiotic Lactobacillus strains in children with atopic dermatitis. J Allergy Clin Immunol
2003;111:389-395.
Rosenfeldt V, Michaelsen KF, Jakobsen M, Larsen CN, Mller PL, Pedersen P, Tvede M,
Weyrehter H, Valerius NH, Paerregaard A. Effect of probiotic Lactobacillus strains in young children
hospitalized with acute diarrhea. Pediatr Infect Dis J 2002;21:411-416.
Rudney JD, Chen R, Sedgewick GJ. Actinobacillus actinomycetemcomitans, Porphyromonas
gingivalis, and Tannerella forsythensis are components of a polymicrobial intracellular flora within
human buccal cells. J Dent Res 2005;84:59-63.
Rudney JD, Krig MA, Neuvar EK, Soberay AH, Iverson L. Antimicrobial proteins in human
unstimulated whole saliva in relation to each other, and to measures of health status, dental plaque
accumulation and composition. Arch Oral Biol 1991;36:497-506.
Rudney JD. Saliva and dental plaque. Adv Dent Res 2000;14:29-39.
Venegas SC, Palacios JM, Apella MC, Morando PJ, Blesa MA. Calcium modulates interactions
between bacteria and hydroxyapatite. J Dent Res 2006;85:1124-1128.
Sakakibara J, Nagano K, Murakami Y, Higuchi N, Nakamura H, Shimozato K, Yoshimura F.
Loss of adherence ability to human gingival epithelial cells in S-layer protein-deficient mutants of
Tannerella forsythensis. Microbiology 2007;153:866-876.
Sakamoto M, Umeda M, Benno Y. Molecular analysis of human oral microbiota. J Periodontal
Res 2005;40:277-285.
Salminen MK, Tynkkynen S, Rautelin H, Poussa T, Saxelin M, Ristola M, Valtonen V,
Jrvinen A. The efficacy and safety of probiotic Lactobacillus rhamnosus GG on prolonged,
noninfectious diarrhea in HIV patients on antiretroviral therapy: a randomized, placebo-controlled,
crossover study. HIV Clin Trials 2004;5:183-191.
Salminen MK. Lactobacillus bacteremia, with special focus on the safety of probiotic
Lactobacillus rhamnosus GG. Academic dissertation, 2006, Helsinki, Finland.
77
Salminen S, Ouwehand A, Benno Y, Lee Y-K. Probiotics: how should they be defined? Trends
Food Sci Technol 1999;10:1-4.
Salo T, Lyons JG, Rahemtulla F, Birkedal-Hansen H, Larjava H. Transforming growth factorbeta 1 up-regulates type IV collagenase expression in cultured human keratinocytes. J Biol Chem
1991;25:11436-11441.
Sanders ME. Probiotics. Food Technol 1999;53:67-77.
Sarker SA, Sultana S, Fuchs GJ, Alan NH, Azim T, Brussow H, Hammarstrm L. Lactobacillus
paracasei strain ST11 has no effect on rotavirus but ameliorates the outcome of nonrotavirus diarrhea
in children from Bangladesh. Pediatrics 2005;116:221-228.
Sasaki M, Bosman BW, Tan PS. Comparison of proteolytic activities in various lactobacilli. J
Dairy Res 1995;62:601-610.
Savaiano DA, ElAnouar A, Smith DE, Levitt MD. Lactose malabsorption from yogurt,
pasteurized yogurt, sweet acidophilus milk, and cultured milk in lactase-deficient individuals. Am J
Clin Nutr 1984;40:1219-1223.
Schaafsma G, Meuling WJ, van Dokkum W, Bouley C. Effects of a milk product, fermented by
Lactobacillus acidophilus and with fructo-oligosaccharides added, on blood lipids in male volunteers.
Eur J Clin Nutr 1998;52:436-440.
Schaffner DW, Beuchat LR. Fermentation of aqueous plant seed extracts by lactic acid bacteria.
Appl Environ Microbiol 1986; 51: 10721076.
Scheie AA, Petersen FC. The biofilm concept: consequences for future prophylaxis of oral
diseases? Crit Rev Oral Biol Med 2004;15: 412.
Schpbach P, Oppenheim FG, Lendenmann U, Lamkin MS, Yao Y, Guggenheim B. Electronmicroscopic demonstration of proline-rich proteins, statherin, and histatins in acquired enamel pellicles
in vitro. Eur J Oral Sci 2001;109:60-68.
Selvam R, Maheswari P, Kavitha P, Ravichandran M, Sas B, Ramchand CN. Effect of Bacillus
subtilis PB6, a natural probiotic on colon mucosal inflammation and plasma cytokines levels in
inflammatory bowel disease. Indian J Biochem Biophys 2009;46:79-85.
Semba RD, Blaum CS, Bartali B, Xue Q-L, Ricks MO, Guralnik JM. Denture use, malnutrition,
frailty and mortality among older women living in the community. J Nutr Health Aging 2006;10:161
167.
Shah NP, Jelen P. Survival of lactic acid bacteria and their lactases under acidic conditions. J
Food Sci 1990;55:506-509.
Shaw J, Nelson L, Hayes C; Duggan C, Watkins J, Walker A (Eds) (2003). Nutrition in
pediatrics: Function and nature of the components in the oral cavity, BC Decker Inc., Ontario, USA.
Shimada T. Salivary proteins as a defense against dietary tannins. J Chem Ecol 2006;32:11491163.
Shimauchi H, Mayanagi G, Nakaya S, Minamibuchi M, Ito Y, Yamaki K, Hirata H.
Improvement of periodontal condition by probiotics with Lactobacillus salivarius WB21: a
randomized, double-blind, placebo-controlled study. J Clin Periodontol 2008;35:897-905.
78
Shimazaki Y, Soh I, Saito T, Yamashita Y, Koga T, Miyazaki H. Influence of dentition status

on physical disability, mental impairment, and mortality in institutionalised elderly people. J Dent Res
2001;80:340345.
Shivakumar KM, Vidya SK, Chandu GN. Dental caries vaccine. Indian J Dent Res 2009;20:99106.
Shortt C. The probiotic century: historical and current perspectives. Trends Food Sci Technol
1999;10:411-417.
Silva N, Dutzan N, Hernandez M, Dezerega A, Rivera O, Aguillon JC, Aravena O, Lasters P,
Pozo P, Vernal R, Gamonal J. Characterization of progressive periodontal lesions in chronic
periodontitis patients: levels of chemokines, cytokines, matrix metalloproteinase-13, periodontal
pathogens and inflammatory cells. J Clin Periodontol 2008;35:206-214.
Slots J. Herpesviruses in periodontal diseases. Periodontol 2000 2005;38:33-62.
Smid E J, Poolman B, Konings W. Casein utilization by lactococci. Appl Environ Microbiol
1991;57:2447-2452
Snydman DR. The safety of probiotics. Clin Infect Dis 2008;46:104-111.
Socransky SS, Haffajee AD, Cugini MA, Smith C, Kent RL, Jr. Microbial complexes in
subgingival plaque. J Clin Periodontol 1998;25:134-144.
Soikkonen K, Wolf J, Salo T, Tilvis R. Radiographic periodontal attachment loss as an indicator
of death risk in the elderly. J Clin Periodontol 2000;27:8792.
Sorsa T, Salo T, Koivunen E, Tyynel J, Konttinen YT, Bergmann U, Tuuttila A, Niemi E,
Teronen O, Heikkil P, Tschesche H, Leinonen J, Osman S, Stenman UH. Activation of type IV
procollagenases by human tumor-associated trypsin-2. J Biol Chem 1997;272:21067-21076.
Splieth CH, Alkilzy M, Schmitt J, Berndt C, Welk A. Effect of xylitol and sorbitol on plaque
acidogenesis. Quintessence Int 2009;40:279-285.
Sreenivasan PK, Meyer DH, Fives-Taylor PM. Requirements for invasion of epithelial cells by
Actinomyces actinomycetemcomitans. Infect Immun 1993;61:1239-1245.
Stefanova M. Bulgarian Yogurt, 1985, ZEMIZDAT, Sofia, Bulgaria p. 29 (in Bulgarian).
Strachinic I, Busarcevic M, Pavlica D, Malasin J, Golic N, Topisirovic L. Molecular and
biochemical characterizations of human oral lactobacilli as putative probiotic candidates. Oral
Teanpaisan R, Dahlen G. Use of polymerase chain reaction techniques and sodium dodecyl
sulfate-polyacrylmide gel electrophoresis for differentiation of oral Lactobacillus species. Oral
Microbil Immunol 2006;21:79-83.
Tellefson LM, Germaine G. Adherence of Streptococcus sanguinis to hydroxyapatite coated
with lysozyme and lysozyme-supplemented saliva. Infect Immun 1986; 51: 750759.
Teng YT. Protective and destructive immunity in the periodontium: Part 1-innate and humoral
immunity and the periodontium. J Dent Res 2006a;85:198-208.
Teng YT. Protective and destructive immunity in the periodontium: Part 2-T-cellmediated
immunity in the periodontium. J Dent Res 2006b;85:209-219.
79
Tenovuo J (Ed) (1989). Human saliva: clinical chemistry and microbiology, Vol. 2. Boca
Raton: CRC Press Inc.
Terahara M, Nishide S, Kaneko T. Preventive effect of Lactobacillus delbrueckii subsp.
bulgaricus on the oxidation of LDL. Biosci Biotechnol Biochem 2000;64:1868-1873.
Teran CG, Teran-Escalera CN, Villarroel P. Nitazoxanide vs. probiotics for the treatment of
acute rotavirus diarrhea in children: a randomized, single-blind, controlled trial in Bolivian children.
Int J Infect Dis 2009;13:518-523.
Teughels W, Newman MG, Coucke W, Haffajee AD, Van der Mei HC, Haake SK, Schepers E,
Cassiman JJ, Van Eldere J, van Steenberge D, Quirynen M. Guided periodontal pocket recolonization:
a proof of concept. J Dent Res 2007;86:1078-1082.
Tharmaraj N, Shah NP. Selective enumeration of Lactobacillus delbrueckii ssp. bulgaricus,
Streptococcus
thermophilus,
Lactobacillus
acidophilus,
Bifidobacteria,
Lactobacillus
casei,
Lactobacillus rhamnosus, and Propionibacteria. J Dairy Sci 2003;86:2288-2296.

Thorstensson H, Johansson B. Does oral health say anything about survival in later life?
Findings in a Swedish cohort of 80+ years at baseline. Community Dent Oral Epidemiol 2009;37:325
332.
Tiranathanagul S, Pattamapun K, Yongchaitrakul T, Pavasant P. MMP-2 activation by
Actinobacillus actinomycetemcomitans supernatant in human PDL cells was corresponded with
reduction of TIMP-2. Oral Dis 2004;10:383-388.
Toba T, Yoshioka E, Iton T. Lacticin, a bacteriocin produced by Lactobacillus delbrueckii
subsp. lactis. Lett Appl Microbiol 1991;12:43-45.
Torriani S, Vescovo M, Dicks MT. Streptococcus thermophilus and Lactobacillus delbrueckii
subsp bulgaricus: a review. Annali di Microb Enzim1997;47:2952.
Trapp CL, Chang CC, Halperen GM, Keen CL, Gershwin ME. The influence of chronic yogurt
consumption on populations of young and elderly adults. Int J Immunother 1993;IX:53-64.
Tukey H B. Leaching of substrates from plants. Annu Rev Plant Physiol 1970;21:305324.
Twetman S, Derawi B, Keller M, Ekstrand K, Yucel-Lindberg T, Stecksen-Blicks K. Shortterm effect of chewing gums containing probiotic Lactobacillus reuteri on the levels of inflammatory
mediators in gingival crevicular fluid. Acta Odontol Scand 2009;67:19-24.
Van de Water J, Keen CL, Gershwin ME. The influence of chronic yogurt consumption on
immunity. J Nutr 1999;129:1492-1495.
van der Ploeg JR. Regulation of bacteriocin production in Streptococcus mutans by the quorumsensing system required for development of genetic competence. J Bacteriol 2005;187:39803989.
Vandamme P, Pot B, Gillis M, De Vos P, Kersters K, Swings J. Polyphasic taxonomy, a
consensus approach to bacterial systematics. Microbiol Rev 1996; 60: 407438.
Vergin F. Antibiotics and probiotics. Hippokrates 1954;25:116-119.
Vesterlund S, Vankerckhoven V, Saxelin M, Goossens H, Salminen S, Ouwehand A. Safety
assessment of Lactobacillus strains: presence of putative risk factors in faecal, blood and probiotic
isolates. Int J Food Microbiol 2007:116:325-331.
80
Vieira LQ, dos Santos LM, Neuman E, da Silva AP, Moura LN, Nicoli JR. Probiotics protect
mice against experimental infections. J Clin Gastroenterol 2008;42:168-169.
Viljanen M, Savilahti E, Haahtela T, Juntunen-Backman K, Korpela R, Poussa T, Tuure T,
Kuitunen M. Probiotics in the treatment of atopic eczema/dermatitis syndrome in infants: a doubleblind placebo controlled trial. Allergy 2005;60:494-500.
Visse R, Nagase H. Matrix metalloproteinases and tissue inhibitors of metalloproteinases:
structure, function and biochemistry. Circ Res 2003;92:827839.
Vitkov L, Krautgartner WD, Hannig M. Bacterial internalization in periodontitis. Oral
Vitorino R, Lobo MJ, Duarte JR, Ferrer-Correia AJ, Domingues PM, Amado FM. The role of
salivary peptides in dental caries. Biomed Chromatogr 2005;19:214-222.
Volozhin AI, Il'in VK, Maksimovski IuM, Sidorenko AB, Istranov LP, Tsarev VN, Istranova
EV, Aboiants RK. Development and use of periodontal dressing of collagen and Lactobacillus casei 37
cell suspension in combined treatment of periodontal disease of inflammatory origin (a microbiological
study). Stomatologiia (Mosk) 2004;83:6-8.
Wada M, Nagata S, Saito M, Shimizu T, Yamashiro Y, Matsuki T, Asahara T, Nomoto K.
Effects of the enteral administration of Bifidobacterium breve on patients undergoing chemotherapy
for pediatric malignancies. Support Care Cancer 2009; (online first).
Wade W. Unculturable bacteria - the uncharacterized organisms that cause oral infections. J R
Soc Med 2002;95:81-83.
Wadstrm T, Andersson K, Sydow M, Axelsson L, Lindgren S, Gullmar B. Surface properties
of lactobacilli isolated from the small intestine of pigs. J Appl Bacter 1987;62:513520.
Wanger RD, Pierson C, Warner T, Dohnalek M, Hilty M, Balish E. Probiotic effects of feeding
heat-killed Lactobacillus acidophilus and Lactobacillus casei to Candida albicans-colonized
immunodeficient mice. J Food Prot 2000;63:638-644.
Wang B, Li J, Li Q, Zhang H, Li N. Isolation of adhesive strains and evaluation of the
colonization and immune response by Lactobacillus plantarum L2 in the rat gastrointestinal tract. Int J
Food Microbiol 2009;132:59-66.
Weiss N, Schillinger U, Kandler O. Lactobacillus lactis, Lactobacillus leichmanii, and
Lactobacillus bulgaricus, subjective synonyms of Lactobacillus delbrueckii subsp. lactis comb. nov.
and Lactobacillus delbrueckii subsp. bulgaricus comb. nov. Syst Appl Microbiol 1984;4:552-557.
West CE, Hammarstrm ML, Hernell O. Probiotics during weaning reduce the incidence of
eczema. Pediatr Allergy Immunol 2009;20:430-437.
Wicht MJ, Haak R, Lummert D, Noack MJ. Treatment of root caries lesions with
chlorhexidine-containing varnishes and dentin sealants. Am J Dent 2003;16:25-30.
Wilson M (Ed) (2005). Microbial inhabitants of humans: Their ecology and role in health and
disease. Cambridge University Press, UK.
Woessner JP. Matrix metalloproteinases and their inhibitors in connective tissue remodeling.
FASEB J 1991;5:21452154.
81
Yamazaki-Kubota T, Miyamoto M, Sano Y, Kusumoto M, Yonezu T, Sugita K, Okuda K,

Yakushiji M, Ishihara K. Analysis of matrix metalloproteinase (MMP-8 and MMP-2) activity in
gingival crevicular fluid from children with Down's syndrome. J Periodontal Res 2009;Sept.
Yee R, McDonald N, Helderman WH. Gains in oral health and improved quality of life of 1213-year-old Nepali schoolchildren: outcomes of an advocacy project to fluoridate toothpaste. Int Dent J
2006;56:196-202.
Yli-Knuuttila H, Snall J, Kari K, Meurman JH. Colonization of Lactobacillus rhamnosus GG in
the oral cavity. Oral Microbil Immunol 2006;21:129-131.
Zeng J, Li Y-Q, Zuo X-L, Zhen Y-B, Yang J, Liu CH. Clinical trial: effect of active lactic acid
bacteria on mucosal barrier function in patients with diarrhoea-predominant irritable bowel syndrome.
Aliment Pharmacol Ther 2008;28:994-1002.
Zhou C, Ma F-Z, Deng X-J, Yuan H, Ma H-S. Lactobacilli inhibit interleukin-8 production
induced by Helicobacter pylori lipopolysaccharide-activated Toll-like receptor 4. World J
Gastroenterol 2008;14:5090-5095.
Zocco MA, dal Verme LZ, Cremonini F, Piscaglia AC, Nista EC, Candelli M, Novi M,
Rigante D, Cazzato IA, Ojetti V, Armuzzi A, Gasbarrini G, Gasbarrini A. Efficacy of Lactobacillus GG
in maintaining remission of ulcerative colitis. Aliment Pharmacol Ther 2006;23:1567-1574.
82

Probioti

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Probioti

Uploaded by

Copyright:

Available Formats

See

Probiotic activity of Lactobacillus delbrueckii

Institute of Dentistry, Biomedicum Research Laboratory,

Probiotic activity of Lactobacillus delbrueckii

Iva Vaseva Stamatova

ISBN 978-952-10-6152-3 (pbk.)

Professor Jukka H. Meurman, MD, PhD, DOdont

Professor Maria Baltadjieva, PhD

Professor Jorma Tenovuo, DDS, PhD

Professor Riita Korpela, PhD

Professor Seppo Salminen, PhD

List of original publications

2 Review of the literature

2.1 Oral cavity in health and disease

Key elements of oral homeostasis

2.2 Common oral diseases

2.2.1.1 Etiology and pathogenesis

2.2.1.2 Treatment and prevention

2.2.2 Periodontal disease

2.2.2.1 Etiology and pathogenesis

2.2.2.2 Treatment and prevention

2.3 Probiotics from the oral health perspective

2.3.1.1 Emergence and definition of the term

2.3.1.2 Beneficial effects of probiotics

2.3.1.3 Selection criteria for probiotic candidates

2.3.1.3.1 Attachment, adhesion, and oral colonization

2.3.1.3.2 Resistance to oral defence mechanisms

2.3.2 Clinical relevance of probiotics in the oral cavity

2.3.2.1 Probiotics and dental caries

2.3.2.2 Probiotics and periodontal disease

2.3.2.3 Probiotics and other oral disorders

2.4 L. delbrueckii subsp. bulgaricus as a probiotic

2.5 Issues of safety in the oral health perspective

3 Hypotheses and aims of the study

4 Materials and methods

4.1 Bacterial strains and culture conditions

4.1.1 Lactic acid bacteria

4.1.2 Oral bacteria

4.1.3 Cell cultures

4.3 Study design

4.3.1 Antimicrobial activity against various oral pathogens (I)

4.3.2 Adhesion to saliva-coated surfaces in vitro (II)

4.3.2.1 Adhesion to sHA beads

4.3.2.2 Adhesion to saliva-coated microtiter plates

4.3.2.3 Adhesion to solvents

4.3.2.4 Effect of lactobacilli pretreatment on streptococcal adhesion in

4.3.3 Proteolytic activity on human gelatinases (III)

4.3.4 Epithelial cell response to L. delbrueckii subsp. bulgaricus

4.3.4.1 Induction of IL-8 and TNF- secretion

4.3.4.2 Epithelial cell response to P. gingivalis after lactobacilli

4.4 Statistical analyses

5 Results and discussion

5.1 Inhibition of oral pathogens (Study I)

5.2 L. bulgaricus adhesion to saliva-coated surfaces (Study II)

5.3 Proteolytic activity on human progelatinase B (proMMP-9) (Study III)

5.3.1 Gelatinolytic activity

5.3.2 Activation of proMMP-9

5.3.2 Proteolytic activity of L. bulgaricus strains in the presence of synthetic

5.4 Epithelial cell lactobacilli interactions (Study IV)

6 Key findings and conclusions

List of original publications

Stamatova I, Kari K, Meurman JH. In vitro evaluation of antimicrobial

Stamatova I, Kari K, Vladimirov S, Meurman JH. In vitro evaluation of